Transgenic mammals having human Ig loci including plural Vh and Vk regions and antibodies produced therefrom

ABSTRACT

The present invention relates to transgenic non-human animals that are engineered to contain human immunoglobulin gene loci. In particular, animals in accordance with the invention possess human Ig loci that include plural variable (V H  and Vκ) gene regions. Advantageously, the inclusion of plural variable region genes enhances the specificity and diversity of human antibodies produced by the animal. Further, the inclusion of such regions enhances and reconstitutes B-cell development to the animals, such that the animals possess abundant mature B-cells secreting extremely high affinity antibodies.

This application is a continuation application of U.S. application Ser.No. 10/078,958, now allowed, which is a continuation application of U.S.application Ser. No. 08/759,620, filed Dec. 3, 1996, abandoned, thedisclosures of which are incorporated herein by their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to transgenic non-human animals that areengineered to contain human immunoglobulin gene loci. In particular,animals in accordance with the invention possess human Ig loci thatinclude plural variable (V_(H) and Vκ) gene regions. Advantageously, theinclusion of plural variable region genes enhances the specificity anddiversity of human antibodies produced by the animal. Further, theinclusion of such regions enhances and reconstitutes B-cell developmentto the animals, such that the animals possess abundant mature B-cellssecreting extremely high affinity antibodies.

2. Background of the Technology

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study of the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs)—an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derivatized Mabs and thus to increase the efficacy andsafety of the administered antibodies. The use of fully human antibodiescan be expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated antibodyadministrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human Mabs with thedesired specificity could be readily produced and selected.

This general strategy was demonstrated in connection with our generationof the first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineeredwith 245 kb and 190 kb-sized germline configuration fragments of thehuman heavy chain loci and kappa light chain loci, respectively, whichcontained core variable and constant region sequences. Id. The human Igcontaining yeast artificial chromosomes (YACs) proved to be compatiblewith the mouse system for both rearrangement and expression ofantibodies, and were capable of substituting for the inactivated mouseIg genes. This was demonstrated by their ability to induce B-celldevelopment and to produce an adult-like human repertoire of fully humanantibodies and to generate antigen-specific human Mabs. These resultsalso suggested that introduction of larger portions of the human Ig locicontaining greater numbers of V genes, additional regulatory elements,and human Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization.

Such approach is further discussed and delineated in U.S. patentapplication Ser. Nos. 07/466,008, filed Jan. 12, 1990, 07/610,515, filedNov. 8, 1990, 07/919,297, filed Jul. 24, 1992, 07/922,649, filed Jul.30, 1992, filed 08/031,801, filed Mar. 15, 1993, 08/112,848, filed Aug.27, 1993, 08/234,145, filed Apr. 28, 1994, 08/376,279, filed Jan. 20,1995, 08/430,938, Apr. 27, 1995, 08/464,584, filed Jun. 5, 1995,08/464,582, filed Jun. 5, 1995, 08/463,191, filed Jun. 5, 1995,08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5, 1995,08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5, 1995,08/462,513, filed Jun. 5, 1995, and 08/724,752, filed Oct. 2, 1996. Seealso European Patent No., EP 0 463 151 B1, grant published Jun. 12,1996, International Patent Application No., WO 94/02602, published Feb.3, 1994, International Patent Application No., WO 96/34096, publishedOct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr. 29,1996. The disclosures of each of the above-cited patents andapplications are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806 and 5,625,825, both to Lonberg and Kay, and GenPharmInternational U.S. patent application Ser. Nos. 07/574,748, filed Aug.29, 1990, 07/575,962, filed Aug. 31, 1990, 07/810,279, filed Dec. 17,1991, 07/853,408, filed Mar. 18, 1992, 07/904,068, filed Jun. 23, 1992,07/990,860, filed Dec. 16, 1992, 08/053,131, filed Apr. 26, 1993,08/096,762, filed Jul. 22, 1993, 08/155,301, filed Nov. 18, 1993,08/161,739, filed Dec. 3, 1993, 08/165,699, filed Dec. 10, 1993,08/209,741, filed Mar. 9, 1994, the disclosures of which are herebyincorporated by reference. See also International Patent ApplicationNos. WO 94/25585, published Nov. 10, 1994, WO 93/12227, published Jun.24, 1993, WO 92/22645, published Dec. 23, 1992, WO 92/03918, publishedMar. 19, 1992, the disclosures of which are hereby incorporated byreference in their entirety. See further Taylor et al., 1992, Chen etal., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al.,(1994), Taylor et al., (1994), and Tuaillon et al., (1995), thedisclosures of which are hereby incorporated by reference in theirentirety.

The inventors of Surani et al., cited above, and assigned to the MedicalResearch Counsel (the “MRC”), produced a transgenic mouse possessing anIg locus through use of the minilocus approach. The inventors on theGenPharm International work, cited above, Lonberg and Kay, following thelead of the present inventors, proposed inactivation of the endogenousmouse Ig locus coupled with substantial duplication of the Surani et al.work.

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. Commensurately, however, a significantdisadvantage of the minilocus approach is that, in theory, insufficientdiversity is introduced through the inclusion of small numbers of V, D,and J genes. Indeed, the published work appears to support this concern.B-cell development and antibody production of animals produced throughuse of the minilocus approach appear stunted. Therefore, the presentinventors have consistently urged introduction of large portions of theIg locus in order to achieve greater diversity and in an effort toreconstitute the immune repertoire of the animals.

Accordingly, it would be desirable to provide transgenic animalscontaining more complete germline sequences and configuration of thehuman Ig locus. It would be additionally desirable to provide such locusagainst a knockout background of endogenous Ig.

SUMMARY OF THE INVENTION

Provided in accordance with the present invention are transgenic animalshaving a near complete human Ig locus, including both a human heavychain locus and a human kappa light chain locus. Preferably, the heavychain locus includes greater than about 20%, more preferably greaterthan about 40%, more preferably greater than about 50%, and even morepreferably greater than about 60% of the human heavy chain variableregion. In connection with the human kappa light chain, preferably, thelocus includes greater than about 20%, more preferably greater thanabout 40%, more preferably greater than about 50%, and even morepreferably greater than about 60% of the human kappa light chainvariable region. Such percentages preferably refer to percentages offunctional variable region genes.

Further, preferably such animals include the entire D_(H) region, theentire J_(H) region, the human mu constant region, and can additionallybe equipped with genes encoding other human constant regions for thegeneration of additional isotypes. Such isotypes can include genesencoding γ₁, γ₂, γ₃, α, ε, β, and other constant region encoding genes.Alternative constant regions can be included on the same transgene,i.e., downstream from the human mu constant region, or, alternatively,such other constant regions can be included on another chromosome. Itwill be appreciated that where such other constant regions are includedon the same chromosome as the chromosome including the human mu constantregion encoding transgene, cis-switching to the other isotype orisotypes can be accomplished. On the other hand, where such otherconstant region is included on a different chromosome from thechromosome containing the mu constant region encoding transgene,trans-switching to the other isotype or isotypes can be accomplished.Such arrangement allows tremendous flexibility in the design andconstruction of mice for the generation of antibodies to a wide array ofantigens.

Preferably, such mice additionally do not produce functional endogenousimmunoglobulins. This is accomplished in a preferred embodiment throughthe inactivation (or knocking out) of endogenous heavy and light chainloci. For example, in a preferred embodiment, the mouse heavy chainJ-region and mouse kappa light chain J-region and C_(κ)-region areinactivated through utilization of homologous recombination vectors thatreplace or delete the region. Such techniques are described in detail inour earlier applications and publications.

Unexpectedly, transgenic mice in accordance with the invention appear topossess an almost entirely reconstituted immune system repertoire. Thisis dramatically demonstrated when four separate mouse strains arecompared: a first strain contains extensive human heavy chain variableregions and human kappa light chain variable regions and encodes only amu isotype, a second strain contains extensive human heavy chainvariable regions and human kappa light chain variable regions andencodes a mu and gamma-2 isotypes, a third strain contains significantlyless human heavy and kappa light chain variable regions, and a fourthstrain contains a double-inactivated mouse Ig locus. The first andsecond strains undergo similar, if not identical, B-cell development,whereas the third strain has a reduced development and maturation ofB-cells, and the fourth strain contains no mature B-cells. Further, itis interesting to note that production of human antibodies in preferenceto mouse antibodies is substantially elevated in mice having a knock-outbackground of endogenous Ig. That is to say that mice that contain ahuman Ig locus and a functionally inactivated endogenous Ig producehuman antibodies at a rate of approximately 100 to 1000 fold asefficiently as mice that contain only a human Ig locus.

Thus, in accordance with a first aspect of the present invention thereis provided a transgenic non-human mammal having a genome, the genomecomprising modifications, the modifications comprising: an inactivatedendogenous immunoglobulin (Ig) locus, such that the mammal would notdisplay normal B-cell development; an inserted human heavy chain Iglocus in substantially germline configuration, the human heavy chain Iglocus comprising a human mu constant region and regulatory and switchsequences thereto, a plurality of human J_(H) genes, a plurality ofhuman D_(H) genes, and a plurality of human V_(H) genes; and an insertedhuman kappa light chain Ig locus in substantially germlineconfiguration, the human kappa light chain Ig locus comprising a humankappa constant region, a plurality of Jκ genes, and a plurality of Vκgenes, wherein the number of V_(H) and Vκ genes inserted are selected tosubstantially restore normal B-cell development in the mammal. In apreferred embodiment, the heavy chain Ig locus comprises a secondconstant region selected from the group consisting of human gamma-1,human gamma-2, human gamma-3, human gamma-4, alpha, delta, and epsilon.In another preferred embodiment, the number of V_(H) genes is greaterthan about 20. In another preferred embodiment, the number of Vκ genesis greater than about 15. In another preferred embodiment, the number ofD_(H) genes is greater than about 25, the number of J_(H) genes isgreater than about 4, the number of V_(H) genes is greater than about20, the number of Jκ genes is greater than about 4, and the number of Vκgenes is greater than about 15. In another preferred embodiment, thenumber of D_(H) genes, the number of J_(H) genes, the number of V_(H)genes, the number of Jκ genes, and the number of Vκ genes are selectedsuch that the Ig loci are capable of encoding greater than about 1×10⁵different functional antibody sequence combinations. In a preferredembodiment, in a population of mammals B-cell function is reconstitutedon average to greater than about 50% as compared to wild type.

In accordance with a second aspect of the present invention there isprovided an improved transgenic non-human mammal having a genome thatcomprises modifications, the modifications rendering the mammal capableof producing human immunoglobulin molecules but substantially incapableof producing functional endogenous immunoglobulin molecules, theimprovement comprising: insertion into the genome of the mammal ofsuffic6ttient human V_(H), D_(H), J_(H), Vκ, and Jκ genes such that themammal is capable encoding greater than about 1×10⁶ different functionalhuman immunoglobulin sequence combinations.

In accordance with a third aspect of the present invention, there isprovided an improved transgenic non-human mammal having a genome thatcomprises modifications, the modifications rendering the mammal capableof producing human immunoglobulin molecules but substantially incapableof producing functional endogenous immunoglobulin molecules, whichmodifications, with respect to the mammal's incapacity to producefunctional endogenous immunoglobulin molecules would not allow themammal to display normal B-cell development, the improvement comprising:insertion into the genome of the mammal of sufficient human V_(H),D_(H), J_(H), Vκ, and Jκ genes such that the mammal is capable ofencoding greater than about 1×10⁶ different functional humanimmunoglobulin sequence combinations and sufficient V_(H) and Vκ genesto substantially restore normal B-cell development in the mammal. In apreferred embodiment, in a population of mammals B-cell function isreconstituted on average to greater than about 50% as compared to wildtype.

In accordance with a fourth aspect of the present invention, there isprovided a transgenic non-human mammal having a genome, the genomecomprising modifications, the modifications comprising: an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; an inactivatedendogenous kappa light chain Ig locus; an inserted human heavy chain Iglocus, the human heavy chain Ig locus comprising a nucleotide sequencesubstantially corresponding to the nucleotide sequence of yH2; and aninserted human kappa light chain Ig locus, the human kappa light chainIg locus comprising a nucleotide sequence substantially corresponding tothe nucleotide sequence of yK2.

In accordance with a fifth aspect of the present invention there isprovided a transgenic non-human mammal having a genome, the genomecomprising modifications, the modifications comprising: an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; an inserted humanheavy chain Ig locus, the human heavy chain Ig locus comprising anucleotide sequence substantially corresponding to the nucleotidesequence of yH2; and an inserted human kappa light chain Ig locus, thehuman kappa light chain Ig locus comprising a nucleotide sequencesubstantially corresponding to the nucleotide sequence of yK2.

In accordance with a sixth aspect of the present invention, there isprovided a transgenic non-human mammal having a genome, the genomecomprising modifications, the modifications comprising: an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; an inactivatedendogenous kappa light chain Ig locus; an inserted human heavy chain Iglocus, the human heavy chain Ig locus comprising a nucleotide sequencesubstantially corresponding to the nucleotide sequence of yH2 withoutthe presence of a human gamma-2 constant region; and an inserted humankappa light chain Ig locus, the human kappa light chain Ig locuscomprising a nucleotide sequence substantially corresponding to thenucleotide sequence of yK2.

In accordance with a seventh aspect of the present invention, there isprovided a transgenic non-human mammal having a genome, the genomecomprising modifications, the modifications comprising: an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; an inserted humanheavy chain Ig locus, the human heavy chain Ig locus comprising anucleotide sequence substantially corresponding to the nucleotidesequence of yH2 without the presence of a human gamma-2 constant region;and an inserted human kappa light chain Ig locus, the human kappa lightchain Ig locus comprising a nucleotide sequence substantiallycorresponding to the nucleotide sequence of yK2.

In accordance with an eighth aspect of the present invention, there isprovided a method for the production of human antibodies, comprising:inoculating any of the mammals of the first through fifth aspects of theinvention discussed above with an antigen; collecting and immortalizinglymphocytic cells to obtain an immortal cell population secreting humanantibodies that specifically bind to the antigen with an affinity ofgreater than 10⁹ M⁻¹; and isolating the antibodies from the immortalcell populations.

In a preferred embodiment, the antigen is IL-8. In another preferredembodiment, the antigen is EGFR. In another preferred embodiment, theantigen is TNF-α.

In accordance with a ninth aspect of the present invention, there isprovided an antibody produced by the method of the sixth aspect of theinvention, including antibodies to IL-8, EGFR, and TNF-α.

In accordance with a tenth aspect of the present invention, there isprovided an improved method for the production of transgenic mice, thetransgenic mice having a genome, the genome comprising modifications,the modifications comprising insertion of a plurality of human variableregions, the improvement comprising: insertion of the human variableregions from a yeast artificial chromosome.

In accordance with an eleventh aspect of the present invention, thereare provided transgenic mice and transgenic offspring therefrom producedthrough use of the improvement of the eighth aspect of the presentinvention.

In accordance with a twelfth aspect of the present invention, there isprovided a transgenic mammal, the transgenic mammal comprising a genome,the genome comprising modifications, the modifications comprising aninserted human heavy chain immunoglobulin transgene, the improvementcomprising: the transgene comprising selected sets of human variableregion genes that enable human-like junctional diversity and human-likecomplementarity determining region 3 (CDR3) lengths. In a preferredembodiment, the human-like junctional diversity comprises averageN-addition lengths of 7.7 bases. In another preferred embodiment, thehuman-like CDR3 lengths comprise between about 2 through about 25residues with an average of about 14 residues.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIGS. 1A-1B are a schematic representation of the reconstructed humanheavy chain and human kappa light chain loci YACs introduced intopreferred mice in accordance with the invention. YACs spanning the humanheavy chain (1H, 2H, 3H, and 4H) and the human kappa light chainproximal (1K, 2K, and 3K) loci were cloned from human-YAC libraries. Thelocations of the different YACs with respect to the human Ig loci(adopted from Cook and Tomlinson, 1995, and Cox et al., 1994), theirsizes, and non-Ig sequences are indicated (not shown to scale). The YACswere recombined into yeast in a two-step procedure (see Materials andMethods) to reconstruct the human heavy and kappa light chain YACs. yH2,the human heavy chain containing YAC, was further retrofitted with ahuman γ₂ gene sequence. yK2, was the human kappa light chain containingYAC. The YAC vector elements: telomere ▴, centromere ●, mammalian (HPRT,Neo) and yeast selectable markers (TRP1, ADE2, LYS2, LEU2, URA3, HIS3)on the YAC vector arms are indicated. V_(H) segments are classified asgenes with open reading frame ●, pseudogenes □, and unsequenced genes ◯.V_(κ) segments are classified as genes with open reading frames ●, andpseudogenes □. The V genes that we have found to be utilized by theXenoMouse II are marked (*) The V_(H) gene region contained on yH2 ismarked by arrows.

FIGS. 2A-2I show a series of Southern Blot analyses andcharacterizations of the human heavy chain YAC, yH2, integrated in EScells and in XenoMouse strains. FIG. 2A-2E show a series of SouthernBlot analyses of EcoRI (FIGS. 2A, 2C) and BamHI (FIGS. 2B, 2D, 2E)digested DNA (2 μg) prepared from the CGM1 immortalized B-lymphoblastcell line derived from the Washington University YAC library source(Brownstein et al., 1989), yH2 YAC (0.5 μg YAC added to 2 μg of 3B1DNA), unmodified E14TG.3B1 (3B1), and yH2-containing ES cell lines: L10,J9.2, L18, L17, and J17. The probes used for blotting were human V_(H)1(FIG. 2A), D_(H) (FIG. 2B) [18 kb fragment in CGM1 lane represents Dsegments on chromosome 16], V_(H)3 (FIG. 2C), Cμ (FIG. 2D) and J_(H)(FIG. 2E). FIG. 2F-2I show a series of Southern Blot analyses of EcoRI(FIG. 2F, 2G) and BamHI (2H, 2I) digested DNA (10 μg) that was preparedfrom the tails of wildtype (WT, 129×B57BL/6J), XM2A-1, and XM2A-2 (2individual offspring) mice or from the parental yH2-containing ES celllines L10 (slightly underloaded relative to other samples), J9.2, andyK2-containing ES cell line J23.1. The probes used were human V_(H)1(FIG. 2F), V_(H)4 (FIG. 2G), human γ-2 (FIG. 2H), and mouse 3′-enhancer(FIG. 2I, the 5 kb band represents the endogenous mouse 3′-enhancerfragment). Fragment sizes of molecular weight markers (in kb) areindicated.

FIGS. 3A-3I show a series of Southern Blot analyses characterizing thehuman kappa light chain YAC, yK2, integrated in ES cells and inXenoMouse 2A Strains. FIG. 3A-E show a series of Southern Blot analysesof EcoRI (FIGS. 3A, 3C, 3D) and BamHI (FIGS. 3B, 3E) digested DNA (2 μg)prepared from CGM1 cell line (Brownstein et al., 1989, supra), yK2 YAC(0.5 μg YAC DNA added to 2 μg of 3B1 DNA), unmodified E14TG.3B1 (3B1),and yK2-containing ES cell lines: J23.1 and J23.7. The probes used werehuman Va (FIG. 3A), Kde (FIG. 3B), V_(κ)II (FIG. 3C), V_(κ)III (FIG.3D), and C_(κ) (FIG. 3E). FIG. 3F-3I show a series of Southern Blotanalyses of EcoRI-digested DNA (2 μg) that was prepared from the tailsof wildtype (WT, 129×B6), XM2A-1, and XM2A-2 (2 individual offspring)mice or from the parental yH2-containing ES cell lines L10 (slightlyunderloaded relative to other samples), J9.2, and yK2-containing ES cellline J23.1. The probes that were used were human V_(κ)I (FIG. 3F),V_(κ)IV (FIG. 3G), V_(κ)VI (FIG. 3H) and 3′-enhancer (FIG. 3I). Fragmentsizes of molecular weight markers (in kb) are indicated.

FIGS. 4A-4T show B-cell reconstitution and surface expression of humanμ, δ, and κ chains on XenoMouse-derived B-cells and shows flow cytometryanalysis of peripheral blood (FIG. 4A-4H) and spleen (FIG. 4I-4T)lymphocytes from wildtype mice (WT), double inactivated mice (DI), andXenoMouse strains 2A-1 and 2A-2 (XM2A-1, XM2A-2). Four-color flowcytometry analysis was carried out using antibodies to theB-cell-specific marker B220 in combination with anti-human μ, δ, κ, ormouse μ, δ, κ, or λ. The percentage of positively-stained cells is shownin each quadrant. Isolation and staining of cells were performed asdescribed in Materials and Methods. Populations of human κ⁺ and mouse λ⁺cells were determined after first gating for B220⁺μ⁺ populations in theindicated region. Populations of μ⁺ and δ⁺ cells were determined afterfirst gating for B220⁺ cells. The percentage of positive cells within aregion or quadrant is indicated. The FACS profiles shown arerepresentative of several experiments performed on each of the strains.

FIG. 5A-5C show that XenoMouse-derived human antibodies block thebinding of their specific antigens to cells. FIG. 5A shows theinhibition of labeled [I¹²⁵] IL-8 binding to human neutrophils by themouse anti-human IL-8 antibody (R&D Systems) (□) and the fully humanMabs D1.1 (♦), K2.2 (●), K4.2 (▴), and K4.3 (▾). The background bindingof labeled [I¹²⁵]IL-8 in the absence of antibody was 2657 cpm. FIG. 5Bshows the inhibition of labeled [I¹²⁵]EGF to its receptors on A431 cellsby mouse anti-human EGFR antibodies 225 and 528 (□, ∇, respectively;Calbiochem) and the fully human antibodies E1.1 (●), E2.4 (▴), E2.5 (▾)and E2.11 (♦). The background binding of [I¹²⁵]EGF in the absence ofantibodies was 1060 cpm. FIG. 5C shows inhibition of labeled [I¹²⁵]TNF-α binding to its receptors on U937 cells by the mouse anti-humanTNF-α antibody (R&D Systems) (□) and fully human Mabs T22.1 (♦), T22.4(●), T22.8 (▴), and T22.9 (▪). The background binding of [I¹²⁵]TNF-α inthe absence of antibody was 4010 cpm. Control human IgG₂ myelomaantibody (

).

FIGS. 6A-6D (SEQ ID NOS 1-29, respectively, in order of appearance)shows repertoire and somatic hypermutation in XenoMouse-derived fullyhuman Mabs. Predicted amino acid sequences of four anti-IL-8 (FIGS. 6A,6B) and four anti-EGFR (FIGS. 6C, 6D) human IgG₂κ Mabs, divided intoCDR1, CDR2 and CDR3 and the constant regions, C_(γ)2 and C_(κ). The Dand J genes of each antibody are indicated. The amino acid substitutionsfrom the germline sequences are indicated in bold letters.

FIG. 7 is a schematic diagram of the human heavy chain genome and thehuman kappa light chain genome.

FIG. 8 is another schematic diagram showing the construction of the yH2(human heavy chain) YAC.

FIG. 9 is another schematic diagram showing the construction of the yK2(human kappa light chain) YAC.

FIG. 10 is another schematic diagram showing the construction of the yK2(human kappa light chain) YAC.

FIGS. 11A-11I show a series of Southern Blot analyses demonstratingintegration intact of the yH2 (human heavy chain) YAC into ES cells andinto the mouse genome. Detailed discussion is provided in connectionwith FIGS. 2A-2I.

FIGS. 12A-12I show a series of Southern Blot analyses demonstratingintegration intact of the yK2 (human kappa light chain) YAC into EScells and into the mouse genome. Detailed discussion is provided inconnection with FIGS. 3A-3I.

FIGS. 13A-13F show B-cell reconstitution and surface expression of humanμ, δ, and κ chains and mouse λ chains on XenoMouse-derived B-cells andshows flow cytometry analysis of peripheral blood. Further details areprovided in connection with FIGS. 4A-4T.

FIG. 14 shows production levels of human antibodies by XenoMouse IIstrains in comparison to murine antibody production by wild type mice.

FIG. 15 is a repertoire analysis of human heavy chain transcriptsexpressed in XenoMouse II strains. The V_(H) nucleotide sequences havebeen assigned SEQ ID NOS 30-41, respectively, in order of appearance.The 10-mer in column N (first instance) has been assigned SEQ ID NO: 42.The 4^(th), 5^(th), 6^(th), 9^(th) and 11^(th) nucleotide sequences incolumn D_(H) have been assigned SEQ ID NOS 43, 44, 45, 46 and 47,respectively. The first and third nucleotide sequences in column N(second instance) have been assigned SEQ ID NOS 48 and 49, respectively.The nucleotide sequences in column J_(H) have been assigned SEQ ID NOS50-61, respectively, in order of appearance.

FIG. 16 is a repertoire analysis of human kappa light chain transcriptsexpressed in XenoMouse II strains. The V_(H) sequences have beenassigned SEQ ID NOS 62-69, respectively, in order of appearance. TheJ_(κ) sequences have been assigned SEQ ID NOS 70-77, respectively, inorder of appearance.

FIG. 17 is another depiction of the diverse utilization of human V_(H)and V_(κ) genes that have been observed as utilized in XenoMouse IIstrains.

FIG. 18 shows the titers of human antibody production in XenoMouse IIstrains.

FIG. 19 is a depiction of gene utilization of anti-IL-8 antibodiesderived from XenoMouse II strains.

FIG. 20 (SEQ ID NOS 1-8, respectively, in order of appearance) showsheavy chain amino acid sequences of anti-IL-8 antibodies derived fromXenoMouse II strains.

FIG. 21 (SEQ ID NOS 9-16, respectively, in order of appearance) showskappa light chain amino acid sequences of anti-IL-8 antibodies derivedfrom XenoMouse II strains.

FIG. 22 shows blockage of IL-8 binding to human neutrophils by humananti-IL-8 antibodies derived from XenoMouse II strains.

FIG. 23 shows inhibition of CD11b expression on human neutrophils byhuman anti-IL-8 antibodies derived from XenoMouse II strains.

FIG. 24 shows inhibition of IL-8 induced calcium influx by humananti-IL-8 antibodies derived from XenoMouse II strains.

FIG. 25 shows inhibition of IL-8 RB/293 chemotaxsis by human anti-IL-8antibodies derived from XenoMouse II strains.

FIG. 26 is a schematic diagram of a rabbit model of human IL-8 inducedskin inflammation.

FIG. 27 shows the inhibition of human IL-8 induced skin inflammation inthe rabbit model of FIG. 26 with human anti-IL-8 antibodies derived fromXenoMouse II strains.

FIG. 28 shows inhibition of angiogenesis of endothelial cells on a ratcorneal pocket model by human anti-IL-8 antibodies derived fromXenoMouse II strains.

FIG. 29 is a depiction of gene utilization of human anti-EGFR antibodiesderived from XenoMouse II strains.

FIG. 30 (SEQ ID NOS 17-23, respectively, in order of appearance) showsheavy chain amino acid sequences of human anti-EGFR antibodies derivedfrom XenoMouse II strains.

FIG. 31 shows blockage EGF binding to A431 cells by human anti-EGFRantibodies derived from XenoMouse II strains.

FIG. 32 shows inhibition of EGF binding to SW948 cells by humananti-EGFR antibodies derived from XenoMouse II strains.

FIG. 33 shows that human anti-EGFR antibodies derived from XenoMouse IIstrains inhibit growth of SW948 cells in vitro.

FIG. 34 shows inhibition of TNF-α binding to U937 cells through use ofhuman anti-TNF-α antibodies derived from XenoMouse II strains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Herein we describe the generation and characterization of severalstrains of mice containing substantially germline configurationmegabase-sized human Ig loci. The present invention thus provides thefirst demonstration of reconstruction of the large and complex human Igloci on YACs and the successful introduction of megabase-sized YACs intomice to functionally replace the corresponding mouse loci.

Mouse Strains

The following mouse strains are described and/or utilized herein:

Double Inactivated (DI) Strain: The DI strain of mice are mice that donot produce functional endogenous, mouse, Ig. In preferred embodiments,the DI mice possess an inactivated mouse J_(H) region and an inactivatedmouse C_(κ) region. The construction of this strain is discussedextensively elsewhere. For example, the techniques utilized forgeneration of the DI strains are described in detail in U.S. patentapplication Ser. Nos. 07/466,008, filed Jan. 12, 1990, 07/610,515, filedNov. 8, 1990, 07/919,297, filed Jul. 24, 1992, 08/031,801, filed Mar.15, 1993, 08/112,848, filed Aug. 27, 1993, 08/234,145, filed Apr. 28,1994, 08/724,752, filed Oct. 2, 1996. See also European Patent No., EP 0463 151 B1, grant published Jun. 12, 1996, International PatentApplication No., WO 94/02602, published Feb. 3, 1994, InternationalPatent Application No., WO 96/34096, published Oct. 31, 1996, and PCTApplication No. PCT/US96/05928, filed Apr. 29, 1996. The disclosures ofeach of the above-cited patent and patent applications are herebyincorporated by reference in their entirety. It has been observed andreported that DI mice possess a very immature B-cell development. Themice do not produce mature B-cells, only pro-B-cells.

XenoMouse I Strain: The design, construction, and analysis of theXenoMouse I strain was discussed in detail in Green et al., NatureGenetics, 7:13-21 (1994). Such mice produced IgMκ antibodies against aDI background. The mice showed improved B-cell function when compared tothe DI strain of mice which have little to no B-cell development. WhileXenoMouse I strains of mice were capable of mounting a sizeable immuneresponse to antigenic challenge, there appeared to be inefficient intheir production of B-cells and possessed a limited response todifferent antigens which apparently was related to their limited V-generepertoire.

L6 Strain: The L6 strain is a mouse producing IgMκ antibodies against aDI background of endogenous mouse Ig. L6 mice contain an inserted humanheavy chain and an inserted human kappa light chain. The L6 strain isgenerated through breeding of a mouse containing a heavy chain insertagainst a double inactivated background (L6H) and a mouse having a kappalight chain insert against a double inactivated background (L6L). Theheavy chain insert comprises an intact approximately 970 kb human DNAinsert from a YAC containing approximately 66 V_(H) segments, startingat V_(H)6-1 and ending at V_(H)3-65, and including the major D geneclusters (approximately 32), J_(H) genes (6), the intronic enhancer(Em), Cμ, and through about 25 kb past Cδ, in germline configuration.The light chain insert comprises an intact approximately 800 kb humanDNA insert from a YAC which contains approximately 32 V_(κ) genesstarting at V_(κ-B3) and ending at V_(κ-Op11). The 800 kb insertcontains a deletion of approximately 100 kb starting at V_(κ-Lp-13) andending at V_(κ-Lp-5). However, the DNA is in germline configuration fromV_(κ-Lp-13) to 100 kb past V_(κ-Op-1), and also contains the J_(κ)genes, the intronic and 3′ enhancers, the constant C_(κ) gene, and Kde.The L6H and L6L mice have been shown to access the full spectrum of thevariable genes incorporated into their genome. It is expected that theL6 mice will similarly access the full spectrum of variable genes intheir genome. Furthermore, L6 mice will exhibit predominant expressionof human kappa light chain, a large population of mature B-cells, andnormal levels of IgM_(κ) human antibodies. Such mice will mount avigorous human antibody response to multiple immunogens, ultimatelyyielding antigen-specific fully human Mabs with subnanomolar affinities.

XenoMouse IIa Strain: The XenoMouse IIa mice represent our secondgeneration XenoMouse™ strains equipped with germline configurationmegabase-sized human Ig loci, against a DI background, such that themice do not produce functional endogenous Ig. Essentially, the mice areequivalent in construction to the L6 strain, but additionally includethe human γ2 gene with its entire switch and regulatory sequences andthe mouse 3′ enhancer in cis. The mice contain an approximately 1020 kbheavy and an approximately 800 kb kappa light chain loci, reconstructedon YACs, which include the majority of the human variable region genes,including heavy chain genes (approximately 66 V_(H)) and kappa lightchain genes (approximately 32 V_(κ)), human heavy constant region genes(μ, δ, and γ) and kappa constant region genes (C_(κ)), and all of themajor identified regulatory elements. These mice have been shown toaccess the full spectrum of the variable genes incorporated into theirgenome. Furthermore, they exhibit efficient class switching and somatichypermutation, predominant expression of human kappa light chain, alarge population of mature B-cells, and normal levels of IgM_(κ) andIgG_(κ) human antibodies. Such mice mount a vigorous human antibodyresponse to multiple immunogens, including human IL-8, human EGFreceptor (EGFR), and human tumor necrosis factor-α (TNF-α), ultimatelyyielding antigen-specific fully human Mabs with subnanomolar affinities.This last result conclusively demonstrates XenoMouse™ as an excellentsource for rapid isolation of high affinity, fully human therapeuticMabs against a broad spectrum of antigens with any desired specificity.

As will be appreciated from the above-introduction, the XenoMouse IIstrain appears to undergo mature B-cell development and mount powerfuladult-human-like immune responses to antigenic challenge. The L6 strain,as predicted from the data in connection with L6L and L6H mice, alsoappear to undergo mature B-cell development and mount powerfuladult-human-like immune responses to antigenic challenge. When DI miceare compared to XenoMouse I strains and DI and XenoMouse I strains arecompared to L6 and XenoMouse II strains, a markedly different B-celldevelopment profile is observed. Owing to this difference, it appearsthat the quantity and/or quality of variable region sequences introducedinto the animals are essential to the induction B-cell maturation anddevelopment and the generation of an adult-human-like immune response.Thus, in addition to the strains' clear use in the generation of humanantibodies, the strains provide a valuable tool for studying the natureof human antibodies in the normal immune response, as well as theabnormal response characteristic of autoimmune disease and otherdisorders.

Variable Region—Quantitative Diversity

It is predicted that the specificity of antibodies (i.e., the ability togenerate antibodies to a wide spectrum of antigens and indeed to a widespectrum of independent epitopes thereon) is dependent upon the variableregion genes on the heavy chain (V_(H)) and kappa light chain (V_(κ))genome. The human heavy chain genome includes approximately 95functional genes which encode variable regions of the human heavy chainof immunoglobulin molecules. In addition, the human light chain genomeincludes approximately 40 genes on its proximal end which encodevariable regions of the human kappa light chain of immunoglobulinmolecules. We have demonstrated that the specificity of antibodies canbe enhanced through the inclusion of a plurality of genes encodingvariable light and heavy chains.

Provided in accordance with the present invention are transgenic micehaving a substantial portion of the human Ig locus, preferably includingboth a human heavy chain locus and a human kappa light chain locus. Inpreferred embodiments, therefore, greater than 10% of the human V_(H)and V_(κ) genes are utilized. More preferably, greater than about 20%,30%, 40%, 50%, 60%, or even 70% or greater of V_(H) and V_(κ) genes areutilized. In a preferred embodiment, constructs including 32 genes onthe proximal region of the V_(κ) light chain genome are utilized and 66genes on the V_(H) portion of the genome are utilized. As will beappreciated, genes may be included either sequentially, i.e., in theorder found in the human genome, or out of sequence, i.e., in an orderother than that found in the human genome, or a combination thereof.Thus, by way of example, an entirely sequential portion of either theV_(H) or V_(κ) genome can be utilized, or various V genes in either theV_(H) or V_(κ) genome can be skipped while maintaining an overallsequential arrangement, or V genes within either the V_(H) or V_(κ)genome can be reordered, and the like. In a preferred embodiment, theentire inserted locus is provided in substantially germlineconfiguration as found in humans. In any case, it is expected and theresults described herein demonstrate that the inclusion of a diversearray of genes from the V_(H) and V_(κ) genome leads to enhancedantibody specificity and ultimately to enhanced antibody affinities.

Further, preferably such mice include the entire D_(H) region, theentire J_(H) region, the human mu constant region, and can additionallybe equipped with other human constant regions for the coding andgeneration of additional isotypes of antibodies. Such isotypes caninclude genes encoding γ₁, γ₂, γ₃, γ₄, α, ε, and δ and other constantregion encoding genes with appropriate switch and regulatory sequences.As will be appreciated, and as discussed in more detail below, a varietyof switch and regulatory sequences can be appropriately utilized inconnection with any particular constant region selection.

The following Table indicates the diversity of antibody combinationsthat are possible in humans, based strictly on random V-D-J joining andcombination with kappa light chains, without consideration of N-additionor somatic mutation events. Based on these considerations, there aregreater than 3.8 million possible antibody combinations in humans, ofany particular isotype. TABLE I Region Heavy Chain Kappa Light ChainVariable “V” ˜95 40 Diversity “D” ≧32 — Joining “J” 6 5 Combinations (V× D × J) 18,240 200 Total Combinations 3.65 × 10⁶ (HC Combinations × LCCombinations)

In connection with a preferred embodiment of the invention, through theinclusion of about 66 V_(H) genes and 32 V_(κ) genes in a mouse with afull complement of D_(H), J_(H), and J_(κ) genes, the possible diversityof antibody production is on the order of 2.03×10⁶ different antibodies.As before, such calculation does not take into account N-addition orsomatic mutation events. Therefore, it will be appreciated that mice inaccordance with the invention, such as the L6 and the XenoMouse IIstrains, offer substantial antibody diversity. In preferred embodiments,mice are designed to have the capability of producing greater than 1×10⁶different heavy chain V-D-J combinations and kappa light chain V-Jcombinations, without accounting for N-additions or somatic mutationevents.

Variable Region—Qualitative Diversity

In addition to quantitative diversity, quantitative selection of V-genes(i.e., large and diverse numbers of V-genes) and/or qualitativeselection of V-genes (i.e., selection of particular V-genes) appears toplay a role in what we refer to herein as “qualitative diversity.”Qualitative diversity, as used herein, refers to diversity in V-D-Jrearrangements wherein junctional diversity and/or somatic mutationevents are introduced. During heavy chain rearrangement, certain enzymes(RAG-1, RAG-2, and possibly others) are responsible for the cutting ofthe DNA representing the coding regions of the antibody genes. Terminaldeoxynucleotidyl transferase (Tdt) activity is upregulated which isresponsible for N-terminal additions of nucleotides between the V-D andD-J gene segments. Similar enzymes and others (SCID and other DNA repairenzymes) are responsible for the deletion that occurs at the junctionsof these coding segments. With respect to junctional diversity, bothN-addition events and formation of the complementarity determiningregion 3 (CDR3) are included within such term. As will be appreciated,CDR3 is located across the D region and includes the V-D and D-Jjunctional events. Thus, N-additions and deletions during both D-Jrearrangement and V-D rearrangement are responsible for CDR3 diversity.

It has been demonstrated that there are certain differences betweenmurine and human junctional diversities. In particular, some researchershave reported that murine N-addition lengths and CDR3 lengths aregenerally shorter than typical human N-addition lengths and CDR3lengths. Such groups have reported that, in humans, N-additions of about7.7 bases in length, on average, are typically observed. Yamada et al.(1991). Mouse-like N-additions are more often on the order of about 3bases in length, on average. Feeney et al. (1990). Similarly, human-likeCDR3 lengths are longer than mouse-like CDR3's. In man CDR3 lengths ofbetween 2 and 25 residues, with an average of 14 residues, is common. Inmice, some groups have reported shorter average CDR3 lengths.

The junctional diversity created by N-additions and CDR3 additions playa clear role developing antibody specificity.

In accordance with the invention, rearranged V-D-J gene sequences showN-addition lengths that are comparable to expected adult-humanN-addition lengths. Further, amino acid sequences across the openreading frame (ORF) corresponding to CDR3 sequences show CDR3 lengthsthat are comparable to expected adult-human CDR3 lengths. Such data isindicative that quantitative variable region diversity and/orqualitative variable region diversity results in human-like junctionaldiversity. Such junctional diversity is expected to lead to a morehuman-like antibody specificity.

Variable Region—Affinities

While we have not conclusively demonstrated a direct causal connectionbetween the increased variable region inclusion and antibodyspecificity, it appears, and it is expected that through providing suchdiversity, the ability of the mouse to mount an immune response to awide array of antigens is possible and enhanced. Additionally, such miceappear more equipped to mount immune responses to a wide array ofepitopes upon individual antigens or immunogens. From our data it alsoappears that antibodies produced in accordance with the presentinvention possess enhanced affinities. Such data includes comparisonsbetween mice in accordance with the invention and the XenoMouse Istrains, as well as consideration of the published results of GenPharmInternational and the MRC. In connection with the XenoMouse I strains,as mentioned above, such mice possessed inefficient B-cell productionand a limited response to different antigens. Such result appearedrelated in part to the limited V-gene repertoire. Similarly, resultsreported by GenPharm International and the MRC indicate a limitedresponse to diverse antigens.

Without wishing to bound to any particular theory or mode of operationof the invention, it would appear that enhanced affinities appear toresult from the provision of the large number of V regions. From ourdata, the provision of greater numbers and/or selection of qualities ofV-gene sequences, enhances junctional diversity (N-additions andformation of complementarity determining region 3 (“CDR3”) diversity),which is typical of an adult-human-like immune response, and which playa substantial role in affinity maturation of antibodies. It may also bethat such antibodies are more effective and efficient in somaticmutation events that lead to enhanced affinities. Each of junctionaldiversity and somatic mutation events are discussed in additional detailbelow.

With respect to affinities, antibody affinity rates and constantsderived through utilization of plural V_(H) and V_(κ) genes (i.e., theuse of 32 genes on the proximal region of the V_(κ) light chain genomeand 66 genes on the V_(H) portion of the genome) results in associationrates (ka in M⁻¹S⁻¹) of greater than about 0.50×10⁻⁶, preferably greaterthan 2.00×10⁻⁶, and more preferably greater than about 4.00×10⁻⁶;dissociation rates (kd in S⁻¹) of greater than about 1.00×10⁻⁴,preferably greater than about 2.00×10⁻⁴, and more preferably greaterthan about 4.00×10⁻⁴; and dissociation constant (in M) of greater thanabout 1.00×10⁻¹⁰, preferably greater than about 2.00×10⁻¹⁰, and morepreferably greater than about 4.00×10⁻¹⁰.

Preferably, such mice additionally do not produce functional endogenousimmunoglobulins. This is accomplished in a preferred embodiment throughthe inactivation (or knocking out) of endogenous heavy and light chainloci. For example, in a preferred embodiment, the mouse heavy chainJ-region and mouse kappa light chain J-region and C_(κ)-region areinactivated through utilization of homologous recombination vectors thatreplace or delete the region.

Variable Region—B-Cell Development

B-cell development is reviewed in Klaus B Lymphocytes (IRL Press (1990))and Chapters 1-3 of Immunoglobulin Genes (Academic Press Ltd. (1989)),the disclosures of which are hereby incorporated by reference.Generally, in mammals, blood cell development, including B- and T-celllymphocytes, originate from a common pluripotent stem cell. Thelymphocytes, then, evolve from a common lymphoid progenitor cell.Following an early gestational period, B-cell initiation shifts from theliver to the bone marrow where it remains throughout the life of themammal.

In the life cycle of a B-cell, the first generally recognizable cell isa pro-pre-B-cell which is found in the bone marrow. Such a cell hasbegun heavy chain V-D-J rearrangement, but does not yet make protein.The cell then evolves into a large, rapidly dividing, pre-B-cell I whichis a cytoplasmically μ⁺ cell. This pre-B-cell I then stops dividing,shrinks, and undergoes light chain V-J rearrangement becoming apre-B-cell II which expresses surface IgM, which leave the marrow asimmature B-cells. Most of the emerging immature B-cells continue todevelop and to produce surface IgD, indicative of their completion ofdifferentiation and development as fully mature immunocompetentperipheral B-cells, which reside primarily in the spleen. However, it ispossible to eliminate the delta constant region and still obtainimmunocompetent cells.

B-cell differentiation and development can be monitored and/or trackedthrough the use of surface markers. For example, the B220 antigen isexpressed in relative abundance on mature B-cells in comparison topre-B-cells I or II. Thus, cells that are B220⁺ and surface IgM⁺ (μ⁺)can be utilized to determine the presence of mature B-cells.Additionally, cells can be screened for surface IgD expression (δ⁺).Another antigen, heat stable antigen, is expressed by pre-B-cells II asthey transition to the periphery (i.e., as they become μ⁺ and/or μ⁺,δ⁺). TABLE II Bone Marrow pre-B-cell II Spleen pro-pre- pre-B-cellemerging immature mature Marker B-cell I B-cell B-cell B-cell B220 − −± + ++ HSA − − + ± − μ − − + + + δ* − − − − +*Assuming the presence of a functional copy of the Cδ gene on thetransgene.

Through use of B-cell markers, such as those mentioned above,development and differentiation of B-cells can be monitored andassessed.

We have previously demonstrated that DI mice (mice that do not undergoheavy chain V-D-J rearrangement or light chain V-J rearrangement) do notproduce mature B-cells. In fact, such mice arrest at the production ofpro-pre-B-cells and B-cells never move from the bone marrow toperipheral tissues, including the spleen. Thus, both B-cell developmentand antibody production are completely arrested. The same result is seenin mice that are only heavy chain inactivated; B-cell development anddifferentiation arrests in the bone marrow.

Our XenoMouse I strain produced functional, somewhat mature B-cells.However, the numbers of B-cells, in both the bone marrow and peripheraltissues, were significantly reduced relative to wild type mice.

In contrast, our XenoMouse II strains and L6 strains, unexpectedlypossess almost complete B-cell reconstitution. Therefore, in accordancewith the invention, we have demonstrated that through the quantitativeinclusion or qualitative inclusion of variable region genes B-celldifferentiation and development can be greatly reconstituted.Reconstitution of B-cell differentiation and development is indicativeof immune system reconstitution. In general, B-cell reconstitution iscompared to wild type controls. Thus, in preferred embodiments of theinvention, populations of mice having inserted human variable regionspossess greater than about 50% B-cell function when compared topopulations of wild type mice.

Further, it is interesting to note that production of human antibodiesin preference to mouse antibodies is substantially elevated in micehaving a knock-out background of endogenous Ig. That is to say that micethat contain a human Ig locus and a functionally inactivated endogenousheavy chain Ig locus produce human antibodies at a rate of approximately100 to 1000 fold as efficiently as mice that only contain a human Iglocus and are not inactivated for the endogenous locus.

Isotype Switching

As is discussed in detail herein, as expected, XenoMouse II mice undergoefficient and effective isotype switching from the human transgeneencoded mu isotype to the transgene encoded gamma-2 isotype. We havealso developed XenoMouse II strains that contain and encode the humangamma-4 constant region. As mentioned above, mice in accordance with theinvention can additionally be equipped with other human constant regionsfor the generation of additional isotypes. Such isotypes can includegenes encoding γ₁, γ₂, γ₃, γ₄, α, ε, δ, and other constant regionencoding genes. Alternative constant regions can be included on the sametransgene, i.e., downstream from the human mu constant region, or,alternatively, such other constant regions can be included on anotherchromosome. It will be appreciated that where such other constantregions are included on the same chromosome as the chromosome includingthe human mu constant region encoding transgene, cis-switching to theother isotype or isotypes can be accomplished. On the other hand, wheresuch other constant region is included on a different chromosome fromthe chromosome containing the mu constant region encoding transgene,trans-switching to the other isotype or isotypes can be accomplished.Such arrangement allows tremendous flexibility in the design andconstruction of mice for the generation of antibodies to a wide array ofantigens.

It will be appreciated that constant regions have known switch andregulatory sequences that they are associated with. All of the murineand human constant region genes had been sequenced and published by1989. See Honjo et al. “Constant Region Genes of the ImmunoglobulinHeavy Chain and the Molecular Mechanism of Class Switching” inImmunoglobulin Genes (Honjo et al. eds., Academic Press (1989)), thedisclosure of which is hereby incorporated by reference. For example, inU.S. patent application Ser. No. 07/574,748, the disclosure of which ishereby incorporated by reference, the cloning of the human gamma-1constant region was prophesized based on known sequence information fromthe prior art. It was set forth that in the unrearranged, unswitchedgene, the entire switch region was included in a sequence beginning lessthan 5 kb from the 5′ end of the first γ-1 constant exon. Therefore theswitch region was also included in the 5′ 5.3 kb HindIII fragment thatwas disclosed in Ellison et al. Nucleic Acids Res. 10:4071-4079 (1982).Similarly, Takahashi et al. Cell 29:671-679 (1982) also reported thatthe fragment disclosed in Ellison contained the switch sequence, andthis fragment together with the 7.7 kb HindIII to BamHI fragment mustinclude all of the sequences necessary for the heavy chain isotypeswitching transgene construction.

Thus, it will be appreciated that any human constant region of choicecan be readily incorporated into mice in accordance with the inventionwithout undue experimentation. Such constant regions can be associatedwith their native switch sequences (i.e., a human γ_(1,2,3, or 4)constant region with a human γ_(1,2,3, or 4) switch, respectively) orcan be associated with other switch sequences (i.e., a human γ₄ constantregion with a human γ₂ switch). Various 3′ enhancer sequences can alsobe utilized, such as mouse, human, or rat, to name a few. Similarlyother regulatory sequences can also be included.

As an alternative to, and/or in addition to, isotype switching in vivo,B-cells can be screened for secretion of “chimeric” antibodies. Forexample, the L6 mice, in addition to producing fully human IgMantibodies, produce antibodies having fully human heavy chain V, D, Jregions coupled to mouse constant regions, such as a variety of gammas(i.e., mouse IgG1,2,3,4) and the like. Such antibodies are highly usefulin their own right. For example, human constant regions can be includedon the antibodies through in vitro isotype switching techniques wellknown in the art. Alternatively, and/or in addition, fragments (i.e.,F(ab) and F(ab′)₂ fragments) of such antibodies can be prepared whichcontain little or no mouse constant regions.

As discussed above, the most critical factor to antibody production isspecificity to a desired antigen or epitope on an antigen. Class of theantibody, thereafter, becomes important according to the therapeuticneed. In other words, will the therapeutic index of an antibody beenhanced by providing a particular isotype or class? Consideration ofthat question raises issues of complement fixation and the like, whichthen drives the selection of the particular class or isotype ofantibody. Gamma constant regions assist in affinity maturation ofantibodies. However, the inclusion of a human gamma constant region on atransgene is not required to achieve such maturation. Rather, theprocess appears to proceed as well in connection with mouse gammaconstant regions which are trans-switched onto the mu encoded transgene.

Materials and Methods

The following Materials and Methods were utilized in connection with thegeneration and characterization of mice in accordance with the presentinvention. Such Materials and Methods are meant to be illustrative andare not limiting to the present invention.

Cloning Human Ig-derived YACs: The Washington University (Brownstein etal., 1989) and the CEPH (Albertsen et al., 1990) human-YAC librarieswere screened for YACs containing sequences from the human heavy andkappa light chain loci as previously described (Mendez et al. 1995).Cloning and characterization of 1H and 1K YACs was described by Mendezet al., (1995). 3H and 4H YACs were identified from the WashingtonUniversity library using a V_(H)3 probe (0.55 kb PstI/NcoI, Berman etal., 1988). The 17H YAC was cloned from the GM1416 YAC library anddetermined to contain 130 kb of heavy chain variable sequences and a 150kb chimeric region at its 3′ end Matsuda et. al., 1993. 2K and 3K YACswere recovered from the CHEF library using V_(κ)II-specific primer(Albertsen et al., 1990).

YAC targeting and recombination: Standard methods foryeast growth,mating, sporulation, and phenotype testing were employed (Sherman et al,1986). Targeting of YAC's and YAC vector arms with yeast and mammalianselectable markers, to facilitate the screening of YAC recombinants inyeast of YAC integration into cells, was achieved by lithium acetatetransformation (Scheistl and Geitz (1989). After every targeting orrecombination step the modified YAC(s) was analyzed by pulsed field gelelectrophoresis and standard Southern Blots to determine the integrityof all sequences.

YAC targeting vectors were used for the interconversion of centric andacentric arms to reorient 17H and to retrofit its 5′ arm with LEU2 andURA3 genes and its 3′ arm with the HIS3 gene. See FIG. 1 a and Mendez etal., 1993. The 4H centric arm was retrofitted with the yeast ADE2 geneand the human HPRT selectable markers. For the first recombination step,a diploid yeast strain was created and selected in which all three YACs17H, 3H, and 4H were present, intact, and stably maintained. A three-wayhomologous recombination between the YAC overlapping regions was inducedby sporulation and the desired recombinant was found by the selection ofthe outer yeast selectable markers (ADE2 and HIS3) and negativeselection (loss) of the internal marker URA3. The successfulrecombination created a 880 kb YAC containing 80% of the IgH variableregion, starting at V_(H)2-5 and extending 20 kb 5′ of the V_(H)3-65gene. For the recombination of the 880 kb YAC to 1H, 1H was retrofittedwith pICL, which adds the LYS2 gene to the centric arm (Hermanson etal., 1991). Using standard yeast mating, a diploid strain was selectedcontaining both 1H and the 880 kb YAC. Upon sporulation and by use ofoverlapping homology, YAC-yeast recombination was carried out. Withpositive selection for the outer yeast markers (ADE2 and URA3) andscreening for the loss of the internal markers (TRP1, LYS2, HIS3), anintact 970 kb YAC consisting of approximately 66 V_(H) segments,starting at V_(H)6-1 and ending at V_(H)3-65 was found. The YAC alsocontained the major D gene clusters, J_(H) genes, the intronic enhancer(Eμ), Cμ, up to 25 kb past Cδ, in germline configuration. This 970 kbYAC was then retrofitted with a targeting vector including a 23 kb EcoRIgenomic fragment of the human γ-2 gene, including its switch andregulatory elements, a 7 kb XbaI fragment of the murine heavy chain 3′enhancer, neomycin gene driven by the metallothionine promoter (MMTNeo),and the yeast LYS2 gene. This vector, while bringing in these sequenceson the 3′ YAC arm, disrupts the URA3 gene.

As a first step toward creating yK2 YAC, by standard yeast mating adiploid yeast strain was selected in which retrofitted 1K and 3K YACswere both present, intact, and stably maintained. Using the same processas described in connection with the IgH construction, YAC-yeastrecombination was carried out. Through use of positive selection for theouter yeast markers (LYS2, TRP1) and the screening for the loss ofinternal markers (URA3, TRP1), an intact 800 kb recombinant product wasfound which contained 32 V_(κ) starting at V_(κ-B3) and ending atV_(κ-Op11). The 800 kb YAC contains a deletion of approximately 100 kbstarting at V_(κ-Lp-13) and ending at V_(κ-Lp-5). However, the YAC is ingermline configuration from V_(κ-Lp-13) to 100 kb past V_(κ-Op-1). TheYAC also contains J_(κ), the intronic and 3′ enhancers, the constantC_(κ), and Kde.

YAC introduction into ES cells and mice: YAC-containing yeastspheroplasts were fused with E14.TG3B1 ES cells as described (Jakobovitset al., 1993a; Green et al., 1994). HAT-resistant colonies were expandedfor analysis. YAC integrity was evaluated by Southern Blot analysisusing protocols and probes described in Berman et al., (1988) and Mendezet al., (1994) and hybridization conditions as described in Gemmil etal., (1991). Chimeric mice were generated by microinjection of ES cellsinto C57BL/6 blastocysts. YAC-containing offspring were identified byPCR analysis of tail DNA as described (Green et al., 1994). YACintegrity was evaluated by Southern Blot analysis using probes andconditions previously described, except that the blot probed with humanV_(H)3 was washed at 50° C.

Flow cytometry analysis: Peripheral blood and spleen lymphocytesobtained from 8-10 week old XenoMice and control mice were purified onLympholyte M (Accurate) and treated with purified anti-mouse CD32/CD16Fc receptor (Pharmingen, 01241D) to block non-specific binding to Fcreceptors, stained with antibodies and analyzed on a FACStar^(PLUS)(Becton Dickinson, CELLQuest software). Antibodies used: allophycocyanin(APC) anti-B220 (Pharmingen, 01129A); biotin anti-human IgM (Pharmingen,08072D); biotin anti-mouse IgM (Pharmingen, 02202D); fluorosceinisothiocyanate (FITC) goat F(ab′)₂ anti-human IgD (SouthernBiotechnology, 2032-02); FITC anti-mouse IgD^(a) (Pharmingen, 05064D);FITC anti-mIgD^(b) (Pharmingen, 05074D); FITC anti-mouse λ (Pharmingen,02174D); PE anti-human κ (Pharmingen, 08175A); PE anti-mouse κ(Pharmingen, 02155A.) RED613™-streptavidin (GibcoBRL, 19541-010) wasused to detect biotinylated antibodies.

Immunization and hybridoma generation: XenoMice (8 to 10 weeks old) wereimmunized intraperitoneally with 25 μg of recombinant human IL-8 or with5 μg TNF-α (Biosource International) emulsified in complete Freund'sadjuvant for the primary immunization and in incomplete Freund'sadjuvant for the additional immunizations carried out at two weekintervals. For EGFR immunization, XenoMice were immunizedintraperitoneally with 2×10⁷ A431 (ATCC CRL-7907) cells resuspended inphosphate buffered saline (PBS). This dose was repeated three times.Four days before fusion, the mice received a final injection of antigenor cells in PBS. Spleen and lymph node lymphocytes from immunized micewere fused with the non-secretory myeloma NSO-bcl2 line (Ray andDiamond, 1994), and were subjected to HAT selection as previouslydescribed (Galfre and Milstein, 1981).

ELISA assay: ELISA for determination of antigen-specific antibodies inmouse serum and in hybridoma supernatants were carried out as described(Coligan et al., 1994) using recombinant human IL-8 and TNF-A andaffinity-purified EGFR from A431 cells (Sigma, E-3641) to capture theantibodies. The concentration of human and mouse immunoglobulins weredetermined using the following capture antibodies: rabbit anti-human IgG(Southern Biotechnology, 6145-01), goat anti-human Igκ (VectorLaboratories, AI-3060), mouse anti-human IgM (CGI/ATCC, HB-57), forhuman γ, κ, and μ Ig, respectively, and goat anti-mouse IgG (Caltag, M30100), goat anti-mouse Igκ (Southern Biotechnology, 1050-01), goatanti-mouse IgM (Southern Biotechnology, 1020-01), and goat anti-mouse λ(Southern Biotechnology, 1060-01) to capture mouse γ, κ, μ, and λ Ig,respectively. The detection antibodies used in ELISA experiments weregoat anti-mouse IgG-HRP (Caltag, M-30107), goat anti-mouse Igκ-HRP(Caltag, M 33007), mouse anti-human IgG2-HRP (Southern Biotechnology,9070-05), mouse anti-human IgM-HRP (Southern Biotechnology, 9020-05),and goat anti-human kappa-biotin (Vector, BA-3060). Standards used forquantitation of human and mouse Ig were: human IgG₂ (Calbiochem,400122), human IgMκ (Cappel, 13000), human IgG₂κ (Calbiochem, 400122),mouse IgGκ (Cappel 55939), mouse IgMκ (Sigma, M-3795), and mouse IgG₃λ(Sigma, M-9019).

Determination of affinity constants of fully human Mabs by BIAcore:Affinity measurement of purified human monoclonal antibodies, Fabfragments, or hybridoma supernatants by plasmon resonance was carriedout using the BIAcore 2000 instrument, using general procedures outlinedby the manufacturers.

Kinetic analysis of the antibodies was carried out using antigensimmobilized onto the sensor surface at a low density: human IL-8-81 RU,soluble EGFR purified from A431 cell membranes (Sigma, E-3641)-303 RU,and TNF-α-107 RU (1,000 RU correspond to about 1 ng/mm² of immobilizedprotein). The dissociation (kd) and association (ka) rates weredetermined using the software provided by the manufacturers,BIAevaluation 2.1.

Affinity measurement by radioimmunoassay: ¹²⁵I-labeled human IL-8(1.5×10⁻¹¹ M or 3×10⁻¹¹ M) was incubated with purified anti-IL-8 humanantibodies at varying concentrations (5×10⁻¹³ M to 4×10⁻⁹ M) in 200 μlof PBS with 0.5% BSA. After 15 hrs. incubation at room temperature, 20μl of Protein A Sepharose CL-4B in PBS (1/1, v/v) was added toprecipitate the antibody-antigen complex. After 2 hrs. incubation at 4°C., the antibody-¹²⁵I-IL-8 complex bound to Protein A Sepharose wasseparated from free ¹²⁵I-IL-8 by filtration using 96-well filtrationplates (Millipore, Cat. No. MADVN65), collected into scintillation vialsand counted. The concentration of bound and free antibodies wascalculated and the binding affinity of the antibodies to the specificantigen was obtained using Scatchart analysis (2).

Receptor binding assays: The IL-8 receptor binding assay was carried outwith human neutrophils prepared either from freshly drawn blood or frombuffy coats as described (Lusti-Marasimhan et al., 1995). Varyingconcentrations of antibodies were incubated with 0.23 nM [¹²⁵I]IL-8(Amersham, IM-249) for 30 min at 4° C. in 96-well Multiscreen filterplates (Millipore, MADV N6550) pretreated with PBS binding buffercontaining 0.1% bovine serum albumin and 0.02% NaN₃ at 25° C. for 2hours. 4×10⁵ neutrophils were added to each well, and the plates wereincubated for 90 min at 4° C. Cells were washed 5 times with 200 μl ofice-cold PBS, which was removed by aspiration. The filters wereair-dried, added to scintillation fluid, and counted in a scintillationcounter. The percentage of specifically bound [¹²⁵I]IL-8 was calculatedas the mean cpm detected in the presence of antibody divided by cpmdetected in the presence of buffer only.

Binding assays for TNF receptor were performed in a similar manner asthe IL-8 assays described above. However, the human monocyte line U937was utilized instead of the neutrophil line used in connection with theIL-8 assays. Antibodies were preincubated with 0.25 nM [¹²⁵]TNF(Amersham, IM-206). 6×10⁵ U937 cells were placed in each well.

The EGF receptor binding assay was carried out with A431 cells (0.4×10⁶cells per well) which were incubated with varying concentrations ofantibodies in PBS binding buffer for 30 minutes at 4° C. 0.1 nM[¹²⁵I]EGF (Amersham, IM-196) was added to each well, and the plates wereincubated for 90 min at 4° C. The plates were washed five times,air-dried and counted in a scintillation counter. Anti-EGFR mouseantibodies 225 and 528 (Calbiochem) were used as controls.

Repertoire analysis of human Ig transcripts expressed in XenoMice andtheir derived human Mabs: Poly(A)⁺ mRNA was isolated from spleen andlymph nodes of unimmunized and immunized XenoMice using a Fast-Track kit(Invitrogen). The generation of random primed cDNA was followed by PCR.Human V_(H) or human V_(κ) family specific variable region primers(Marks et. al., 1991) or a universal human V_(H) primer, MG-30(CAGGTGCAGCTGGAGCAGTCIGG) (SEQ ID NO: 78) was used in conjunction withprimers specific for the human Cμ (hμP2) or Cκ (hκP2) constant regionsas previously described (Green et al., 1994), or the human γ2 constantregion MG-40d; 5′-GCTGAGGGAGTAGAGTCCTGAGGA-3′ (SEQ ID NO: 79). PCRproducts were cloned into pCRII using a TA cloning kit (Invitrogen) andboth strands were sequenced using Prism dye-terminator sequencing kitsand an ABI 377 sequencing machine. Sequences of human Mabs-derived heavyand kappa chain transcripts were obtained by direct sequencing of PCRproducts generated from poly(A⁺) RNA using the primers described above.All sequences were analyzed by alignments to the “V BASE sequencedirectory” (Tomlinson et al., MRC Centre for Protein Engineering,Cambridge, UK) using MacVector and Geneworks software programs.

Preparation and purification of antibody Fab fragments: Antibody Fabfragments were produced by using immobilized papain (Pierce). The Fabfragments were purified with a two step chromatographic scheme: HiTrap(Bio-Rad) Protein A column to capture Fc fragments and any undigestedantibody, followed by elution of the Fab fragments retained in theflow-through on strong cation exchange column (PerSeptive Biosystems),with a linear salt gradient to 0.5 M NaCl. Fab fragments werecharacterized by SDS-PAGE and MALDI-TOF MS under reducing andnon-reducing conditions, demonstrating the expected ˜50 kD unreducedfragment and ˜25 kDa reduced doublet. This result demonstrates theintact light chain and the cleaved heavy chain. MS under reducingconditions permitted the unambiguous identification of both the lightand cleaved heavy chains since the light chain mass can be preciselydetermined by reducing the whole undigested antibody.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Reconstruction of Human Heavy Chain Loci on YACs

In accordance with the present invention, the strategy that we utilizedto reconstruct the human heavy chain and human kappa light chainvariable regions was to, first, screen human-YAC libraries for YACs thatspanned the large (megabase-sized) human Ig loci and, second, torecombine YACs spanning such regions into single YACs containing thedesired loci predominantly in germline configuration.

The above, stepwise, YAC recombination scheme exploited the highfrequency of meiotic-induced homologous recombination in yeast and theability to select the desired recombinants by the yeast markers presenton the vector arms of the recombined YACs (See FIG. 1, and Green et al.,supra.; see also Silverman et al., 1990 and denDunnen et al., 1992).

In connection with our strategy, we identified four YACs, 1H (240 kb),2H (270 kb), 3H (300 kb), and 4H (340 kb), which spanned about 830 kb,out of the about 1000 kb, of the human heavy chain variable region onchromosome 14q. YACs 1H, 2H, 3H, and 4H were used for reconstruction ofthe locus (See FIG. 1A). Pulsed Field Gel Electrophoresis (PFGE) andSouthern blot analysis confirmed the YACs to be in intact, germlineconfiguration, with the exception of 150 kb at the 3′ end of YAC 2Hwhich contained certain non-IgH sequences (See FIG. 1; Matsuda et al.,1990). YAC 1H, the YAC that was previously introduced into our firstgeneration XenoMouse™ (Green et al., supra.; Mendez et al., 1995), iscomprised of the human C_(δ), C_(μ), J_(H), and D_(H) regions and thefirst 5 V_(H) genes in germline configuration. The other three YACscover the majority of the V_(H) region, from V_(H)2-5 to V_(H)3-65, thuscontributing approximately an additional 61 different V_(H) genes. Priorto recombination, YAC 4H was retrofitted with an HPRT selectable marker.Through utilization of the overlapping sequences contained on the YACs,the four YACs (1H, 2H, 3H, and 4H) were recombined in yeast by astepwise recombination strategy (See FIG. 1A). Such recombinationstrategy generated a 980 kb recombinant YAC (See FIG. 1). Analysis ofthe YAC by PFGE and Southern blot analysis confirmed the presence of thehuman heavy chain locus from the C_(δ) region to 20 kb 5′ of theV_(H)3-65 gene in germline configuration. No apparent deletions orrearrangements were observed.

The YAC acentric arm was targeted with a vector bearing the completehuman γ2 constant region, mouse 3′ enhancer, and the neomycin resistancegene, to yield the final 1020 kb heavy chain YAC, yH2. YAC yH2 containedthe majority of the human variable region i.e., 66 out of the 82 V_(H)genes, complete D_(H) (32 genes), and J_(H) (6 genes) regions and threedifferent constant regions (Cμ, Cδ, and Cγ) with their correspondingregulatory sequences (See FIG. 1A). This was the heavy chain constructutilized for the production of our XenoMouse II strains.

Example 2 Reconstruction of Human Kappa Light Chain Loci on YACs

A similar stepwise recombination strategy was utilized forreconstruction of the human kappa light chain locus. Three YACs wereidentified that spanned the human kappa loci. The YACs were designated1K, 2K and 3K. YAC 1K, which had a length of approximately 180 kb, hadpreviously been introduced into our first generation XenoMouse™. SuchYAC contained the kappa deleting element, (Kde), the kappa 3′ andintronic enhancers, C_(κ), J_(κ), and the three V_(κ) genes on the Bcluster (Green et al., 1994; Mendez et al., 1995). YAC 2K (approximately480 kb), and 3K (approximately 380 kb) together encompass most of thekappa chain proximal variable region on chromosome 2p. A deletion ofapproximately 100 kb spans the L13-L5 region (FIG. 1B; Huber et al.,1993). Inasmuch as the kappa distal region duplicates the proximalregion, and as the proximal V_(κ) genes are the ones most commonlyutilized humans (Weichold et al., 1993; Cox et al., 1994), the proximalregion was the focus of our reconstruction strategy (FIG. 1B). Throughhomologous recombination of the three YACS, an 800 kb recombinant YAC,yK2, was recovered. The size and integrity of the recombinant YAC wasconfirmed by PFGE and Southern blot analysis. Such analysis demonstratedthat it covered the proximal part of the human kappa chain locus, with32 V_(κ) genes in germline configuration except for the describeddeletion in the Lp region (FIG. 1B). yK2 centric and acentric arms weremodified to contain the HPRT and neomycin selectable markers,respectively, as described (Materials and Methods). This was the kappalight chain construct utilized for the production of our XenoMouse IIstrains.

The YACs described herein, yH2 and yK2, represent the firstmegabase-sized reconstructed human Ig loci to contain the majority ofthe human antibody repertoire, predominantly in germline configuration.This accomplishment further confirmed homologous recombination in yeastas a powerful approach for successful reconstruction of large, complex,and unstable loci. The selection of stable YAC recombinants containinglarge portions of the Ig loci in yeast provided us with the human Igfragments required to equip the mice with the human antibody repertoire,constant regions, and regulatory elements needed to reproduce humanantibody response in mice.

Example 3 Introduction of yH2 and yK2 YACs into ES Cells

In accordance with our strategy, we introduced the YACs, yH2 and yK2,into mouse embryonic stem (ES) cells. Once ES cells containing the YACDNA were isolated, such ES cells were utilized for the generation ofmice through appropriate breeding.

In this experiment, therefore, YACs yH2 and yK2, were introduced into EScells via fusion of YAC-containing yeast spheroplasts withHPRT-deficient E14.TG3B1 mouse ES cells as previously described(Jakobovits et al., 1993a; Green et al., 1994). HPRT-positive ES cellclones were selected at a frequency of 1 clone/15-20×10⁶ fused cells andwere analyzed for YAC integrity by Southern and CHEF blot analyses(FIGS. 2A-2E).

Seven of thirty-five ES cell clones (referred to as L10, J9.2, L17, L18,J17, L22, L23) derived from ES cell fusion with yH2-containing yeastwere found to contain all expected EcoRI and BamHI yH2 fragmentsdetected by probes spanning the entire insert: mouse 3′ enhancer, humanintronic enhancer, human C_(γ)2, C_(δ), and C_(μ) constant regions,D_(H), J_(H) and all the different V_(H) families: V_(H)1, V_(H)2,V_(H)3, V_(H)4, V_(H)5, and V_(H)6 (data shown for 5 clones in FIGS.2A-2E). CHEF analysis further confirmed that these clones, whichrepresent 20% of all clones analyzed, contain the entire intact yH2 YACwith no apparent deletions or rearrangements (data not shown).

ES cell clones derived from the fusion of yK2-containing yeast weresimilarly analyzed for YAC integrity, using probes specific for thehuman Kde, kappa 3′ and intronic enhancers, C_(κ), J_(κ), and all of thedifferent V_(κ) families: V_(κ)I, V_(κ)II, V_(κ)III, V_(κ)IV, V_(κ)VI.Twenty clones of the sixty clones had intact and unaltered YAC, whichrepresent 30% of total clones analyzed (data shown for two ES clones inFIGS. 3A-3E). Varying amounts of yeast genomic sequences were detectedin yH2 and yK2-ES cell clones (data not shown).

These results are the first demonstration of introduction ofmegabase-sized constructs encompassing reconstructed human loci,predominantly in germline configuration, into mammalian cells. Therelatively high frequency of intact YACs integrated into the mousegenome further validated the ES cell-yeast spheroplast fusionmethodology as an effective approach for faithful introduction of largehuman genomic fragments into ES cells.

Example 4 Generation of XenoMouse II Strains

In order to generate mice from the YAC DNA containing ES cells,microinjection of blastocysts was conducted, followed by breeding. Thus,yH2- and yK2-bearing ES cell clones were expanded and microinjected intomouse C57BL/6J blastocysts (Green et al., 1994) and the chimeric malesproduced were evaluated for germline transmission. Offspring withtransmitted YAC were identified by PCR analysis and the YAC integritywas confirmed by Southern blot analysis. In all transgenic mice analyzedthe YAC was shown to be in intact form (FIGS. 2F-2I, 3F-3I). All sevenmicroinjected yH2-ES clones and two out of eight yK2-ES clones weretransmitted through the mouse germline.

In order to generate mice that produced human antibodies to theexclusion of endogenous antibodies, yH2- or yK2-transgenic mice werebred with double-inactivated (DI) mouse strains. The DI mouse strainsare homozygous for gene targeted-inactivated mouse heavy and kappa chainloci and thus are deficient in antibody production (Jakobovits et al.,1993b; Green et al., 1994). Two of the yH2-transgenic mouse strains L10and J9.2, and one of the yK2-transgenic mouse strains, J23.1, were bredwith DI mice to generate mice bearing YACs on an homozygous inactivatedmouse heavy and kappa chain background (yH2;DI, and yK2;DI). Each of theyH2;DI transgenic strains were bred with the yK2;DI transgenic strain togenerate two XenoMouse II strains, 2A-1 (L10;J23.1;DI) and 2A-2(J9.2;J23.1;DI), respectively, containing both heavy and light chainYACs on homozygous DI background. L10 is fully homozygous and J9.2 andJ23.1 are in the process of being successfully bred to homozygosity.

The integrity of the human heavy and kappa chain YACs in XenoMouse IIstrains was confirmed by Southern blot analysis. As shown in FIG. 2 andFIG. 3, in both XenoMouse strains analyzed, yH2 and yK2 were transmittedunaltered through multiple generations with no apparent deletions orrearrangements.

Example 5 B-Cell Development and Human Antibody Production by XenoMouseII Mice

In order to further characterize the XenoMouse II strains, we studiedtheir B-cell development and their production of human antibodies.Reconstitution of B-cell development and antibody production inXenoMouse II strains by yH2 and yK2 YACs was evaluated by flow cytometryand ELISA. In contrast to DI mice, which completely lack mature B-cells,XenoMouse II manifested essentially normal B-cell development with themature B-cell population in the blood totaling over 50% of the levelseen in wild type mice (FIGS. 4A-4H). All B-cells were shown to expresshuman IgM and high levels of B220 (human IgM⁺/B220^(hi)), with 60% ofthis population also expressing human IgD. Similar results were obtainedfrom analysis of XenoMouse spleen and lymph nodes (not shown). Theseresults correlate well with the characteristics of mature B-cells inwild type mice, indicating proper B-cell maturation in XenoMouse.

The majority of XenoMouse B-cells (75-80%) expressed exclusively humankappa (κ) light chain, whereas only about 15% expressed mouse lambda (λ)light chain (FIGS. 4A-41). This light chain distribution ratio (hκ/mλ:75:15) is comparable to that observed in wild type mice, indicating amouse-like regulation of light chain utilization. In contrast, XenoMouseI, as described in Green et al., 1994, showed a ratio of hκ/mλ: 55:45(data not shown). Similar observations were made for B-cells from spleen(FIGS. 4I-4T) and lymph nodes (not shown), indicating that most ofXenoMouse II's B-cells produced exclusively fully human antibodies.Levels of mλ-expressing B-cells were reduced from 15% to 7% in XenoMouseII strains homozygous for yK2 (data not shown).

Example 6 Generation of L6 Strain

The L6 strain of mice were generated identically to the processdescribed above in connection with the generation of the XenoMouse IIstrains. However, owing to a deletion event during the generation of theL6 ES cell line, the ES cell line, and, subsequently, the L6 mouseevolved without a portion of the sequence distal to Cδ, thus,eliminating the Cγ constant region and its regulatory sequences.Following completion of breeding, the L6 mice will contain the entireyK2 construct and the entire yH2 construct, except for the missing Cγconstant region.

Example 7 Human Antibody Production

Expression of human Cμ, Cγ2, and κ, light chains were detected inunimmunized XenoMouse II sera at maximal levels of 700, 600, and 800μg/ml, respectively. To determine how these values compared towild-type, we measured maximal levels of mouse Cμ, Cγ2, and κ, lightchains in C57BL/6J×129 mice kept under similar pathogen-free conditions.The values for Cμ, Cγ2, and κ light chain in wild-type mice were 400,2000, and 2000 μg/ml, respectively. Upon immunization, the human γ chainlevels increased to approximately 2.5 mg/ml. The concentration of mouseλ was only 70 μg/ml, further confirming the preferential use of humankappa chain.

These findings confirmed the ability of the introduced human Ig YACs toinduce proper Ig gene rearrangement and class switching and to generatesignificant levels of fully human IgM and IgG antibodies before andafter immunization.

Example 8 A Diverse Human Antibody Repertoire in XenoMouse II

In order to further understand the reconstitution of the antibodyrepertoire in XenoMouse II strains, we challenged mice with severalantigens, and prepared hybridoma cell lines secreting such antibodies.As will be understood, recapitulation of the human antibody response inmice requires diverse utilization of the different human variable genescontained on yH2 and yK2 YACs. The diversity of the human antibodiesgenerated by XenoMouse II strains was determined by cloning andsequencing human heavy chain (μ and γ) and kappa light chain transcriptsfrom XenoMouse lymph nodes. Based upon our data to date, sequenceanalysis demonstrates that XenoMouse II utilizes at least 11 out of the37 functional V_(H) genes present on yH2, eight different D_(H) segmentsand three J_(H) genes (J_(H3), J_(H4), J_(H6)) (Table III; J_(H5) wasalso detected in connection with our sequencing antibodies fromhybridomas). V-D-J sequences were linked to human μ or γ2 constantregions (not shown).

The V_(H) genes utilized are widely distributed over the entire variableregion and represent four out of the seven V_(H) families (Table III).The predominant utilization of V genes from V_(H3) and V_(H4) familiesis similar to the V_(H) usage pattern in adult humans, which isproportional to family size (Yamada et al. 1991; Brezinshek et al.,1995). The predominant usage of J_(H4) is also reminiscent of thatdetected in human B-cells (Brezinshek et al., 1995). Addition ofnon-germline nucleotides (N-additions) at both V-D and D-J joinings,ranging from 1-12 bp, were also observed. Such N-additions producedcomplementary determining regions 3 (CDR3s) with lengths of from 8 toabout 19 amino acid residues, which is very comparable to that observedin adults human B-cells (Yamada et al. 1991; Brezinshek et al., 1995).Such CDR3 lengths observed in the XenoMouse II are much longer than CDR3lengths ordinarily observed in mice (Feeny, 1990).

A highly diverse repertoire was also found in the ten kappa chaintranscripts sequenced. In addition to displaying 8 out of the 25 Vκfunctional open reading frames (ORFs) present on yK2, all of the Jκgenes were detectable (Table IV). The different Vκ genes utilized werewidely dispersed throughout yK2, representing all four major Vκ genefamilies. All VκJκ recombination products were linked properly to Cκsequences. The paucity of N-additions in our transcripts is in agreementwith the greatly reduced terminal deoxynucleotide transferase activityat the stage of kappa chain rearrangement. The average CDR3 length of9-10 amino acids that we observed in the kappa chain transcripts isidentical to that observed in human B-cells (Marks et al., 1991).

In Tables III and IV below, repertoire analyses of human heavy and kappalight chain transcripts expressed in XenoMouse II strains are presented.Human μ, γ, and κ specific mRNAs were amplified by PCR, cloned andanalyzed by sequencing as described in Materials and Methods. Table IIIshows a series of nucleotide sequences of 12 unique human heavy chainclones, divided into V_(H), D, J_(H) and N segments, as identified byhomology with published germline sequences (Materials and Methods). EachD segment assignment is based on at least 8 bases of homology. Table IVshows a series of nucleotide sequences of V-J junctions of 8 independenthuman κ clones. The sequences are divided into V_(κ) J_(κ), and Nsegments and identified based on homology to published V_(κ) and J_(κ)sequences. In each of the Tables N-additions and deletions (indicated as_) were determined by their lack of sequence homology to V, D, or Jsequences. TABLE III Repertoire Analysis of Human Heavy ChainTranscripts Clone V_(H) N D_(H) N H_(H) A2.2.1 5-51 (DP73)  4 XP5rc 12JH4_GACTACTGGGGC TTACTGTGCGAGACA (TAGG) AATCAT (GGGAGCTACGGG) (SEQ IDNO: 50) (SEQ ID NO: 30) (SEQ ID NO: 48) B2.1.5 3-33 (DP-50)  7 3rc  7JH4_CTTTGACTACTGGGGC TTACTGTGCGAGAGA (TCGGGGA) AATAGCA (CTGGCCT) (SEQ IDNO: 51) (SEQ ID NO: 31) B4.2.4 3-15 (DP-38)  1 K1 11JH6_CTACTACTACTACGGT TTACTGTACCACAGA (G) GGCTAC (ACTAACTACCC) (SEQ IDNO: 52) (SEQ ID NO: 32) (SEQ ID NO: 49) B4.2.5 4-59 (DP-71) 10 4  6JH6_ACTACTACTACTACGGT TTACTGTGCGAGAGA (TAGGAGTGTT) GTACTACCAGCTGCTAT(ACCCAA) (SEQ ID NO: 53) (SEQ ID NO: 33) (SEQ ID NO: 42) (SEQ ID NO: 43)D2.2.5 4-34 (DP-63)  2 N1 GCAGCAGCTG  4 JH4_CTTTGACTACTGGGGCTTACTGTGCGAGAG_(—) (GG) (SEQ ID NO: 44) (CCCT) (SEQ ID NO: 54) (SEQ IDNO: 34) D2.1.3 3-48 (DP-51)  4 XP1  2 JH6_CTACTACTACTACGGTTTACTGTGCGAGACA (TCTT) GATATTTTGACTGGT (CT) (SEQ ID NO: 55) (SEQ ID NO:35) (SEQ ID NO: 45) D2.2.8 4-31 (DP-65)  2 A4  5 JH4_TTTGACTACTGGGGCTTACTGTGCGAGAGA (GA) GACTGCAG (CGGTT) (SEQ ID NO: 56) (SEQ ID NO: 36)A2.2.4 3-21 (DP-77)  2 IR3  3 JH6_TACTACTACTACTACGGT TTACTGTGCGAGAGA(TT) GGGGCTGG (ACC) (SEQ ID NO: 57) (SEQ ID NO: 37) D4.2.11 4-4/4.35  1N1  2 JH4_CTTTGACTACTGGGGC ATTACTGTGCGA (A) TATAGCAGTGGCTGGT (GT) (SEQID NO: 58) (SEQ ID NO: 38) (SEQ ID NO: 46) C1.2.1 1-18 (DP-14)  0 XP′  0JH4_GACTACTGGGGC TATTACTGTGCGAG_(—) 1/21-7 GTTA (SEQ UD NO: 59) (SEQ IDNO: 39) C3.1.2 4-39 (DP-79)  3 2 GGATATAGTAGTGG  6 JH4_CTTTGACTACTGGGGCTATTACTGTGCG_(—) (GCC) (SEQ ID NO: 47) (TCGGGC) (SEQ ID NO: 60) (SEQ IDNO: 40) D2.2.7 5-51 (DP73)  4 K1  9 JH3 ATGCTTTGATATCTGGGGTTACTGTGCGAGACA (TGGC) AGTGGCT (GGTACTCTG) (SEQ ID NO: 61) (SEQ ID NO:41)

TABLE IV Repertoire Analysis of Human Kappa Light Chain TranscriptsClone Vκ N Jκ F2.2.3 02 (DPK9) 0 Jκ5 TTAAACGAACAGTACCCC_(—)GATCACCTTCGGCCAA (SEQ ID NO: 62) (SEQ ID NO: 70) F4.1.8 L5 (DPK5) 0 Jκ1GGACGTTCGGCCAA ACAGGCTAACAOTTTCCCTC_(—) (SEQ ID NO: 71) (SEQ ID NO: 63)F4.1.6 A20 (DPK4) 0 Jκ3 AAGTATAACAGTGCCCC ATTCACTTTCGGCCCT (SEQ ID NO:63) (SEQ ID NO: 72) F2.2.5 08 0 Jκ4 ACAGTATGATAATCTCCC_(—)GCTCACTTTCGGCGGA (SEQ ID NO: 65) (SEQ ID NO: 73) F2.1.5 L1 0 Jκ5AAAGTATAATAGTTACCC_(—) GATCACCTTCGGCCAA (SEQ ID NO: 66) (SEQ ID NO: 74)F2.1.4 A30 0 Jκ3 CAGCATAATAGTTACCC_(—) ATTCACTTTCGGCCCT (SEQ ID NO: 67)(SEQ ID NO: 75) F2.1.3 B3 (DPK24) 0 Jκ4 AATATTATAGTACTCC_(—)GCTCACTTTCGGCGGA (SEQ ID NO: 68) (SEQ ID NO: 76) F4.1.3 A27 (DPK22) 1Jκ2 CAGTATGGTAGCTCACCTC_(—) (G) _CACTTTTGGCCAG (SEQ ID NO: 69) (SEQ IDNO: 77)

These results, together with sequences of XenoMouse-derived hybridomasdescribed later, demonstrate a highly diverse, adult human-likeutilization of V, D, and J genes, which appears to demonstrate that theentire human heavy and kappa chain variable regions present on the yH2and the yK2 YACs are accessible to the mouse system for antibodyrearrangement and are being utilized in a non-position-biased manner. Inaddition, the average length of N-additions and CDR3s for both the heavyand kappa chain transcripts, is very similar to that seen in adult humanB-cells, indicating that the YAC DNA contained in the mice direct themouse machinery to produce an adult human-like immune repertoire inmice.

In connection with the following Examples, we prepared high affinityantibodies to several antigens. In particular, antigens were prepared tohuman IL-8 and human EGFR. The rationale for the selection of IL-8 andEGFR is as follows.

IL-8 is a member of the C-X-C chemokine family. IL-8 acts as the primarychemoattractant for neutrophils implicated in many diseases, includingARDS, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, psoriasis, alcoholic hepatitis, reperfusion injury,to name a few. Moreover, IL-8 is a potent angiogenic factor forendothelial cells. In FIGS. 22-28, we demonstrate that human anti-IL-8antibodies derived from XenoMouse II strains are effective in ainhibiting IL-8's actions in a number of pathways. For example, FIG. 22shows blockage of IL-8 binding to human neutrophils by human anti-IL-8.FIG. 23 shows inhibition of CD11b expression on human neutrophils byhuman anti-IL-8. FIG. 24 shows inhibition of IL-8 induced calcium influxby human anti-IL-8 antibodies. FIG. 25 shows inhibition of IL-8 RB/293chemotaxsis by human anti-IL-8 antibodies. FIG. 26 is a schematicdiagram of a rabbit model of human IL-8 induced skin inflammation. FIG.27 shows the inhibition of human IL-8 induced skin inflammation in therabbit model of FIG. 26 with human anti-IL-8 antibodies. FIG. 28 showsinhibition of angiogenesis of endothelial cells on a rat corneal pocketmodel by human anti-IL-8 antibodies.

EGFR is viewed as an anti-cancer target. For example, EGFR isoverexpressed, up to 100 fold, on a variety of cancer cells. Ligand (EGFand TNF) mediated growth stimulation plays a critical role in theinitiation and progression of certain tumors. In this regard, EGFRantibodies inhibit ligand binding and lead to the arrest of tumor cellgrowth, and, in conjunction with chemotherapeutic agents, inducesapoptosis. Indeed, it has been demonstrated that a combination of EGFRMabs resulted in tumor eradication in murine xenogeneic tumor models.Imclone has conducted Phase I clinical utilizing a chimeric Mab (C225)that proved to be safe. In FIGS. 31-33, we demonstrate data related toour human anti-EGFR antibodies. FIG. 30 shows heavy chain amino acidsequences of human anti-EGFR antibodies derived from XenoMouse IIstrains. FIG. 31 shows blockage EGF binding to A431 cells by humananti-EGFR antibodies. FIG. 32 shows inhibition of EGF binding to SW948cells by human anti-EGFR antibodies. FIG. 33 shows that human anti-EGFRantibodies derived from XenoMouse II strains inhibit growth of SW948cells in vitro.

Example 9 High Affinity, Antigen-Specific Human Mabs Produced byXenomouse II

We next asked whether the demonstrated utilization of the large humanrepertoire in XenoMouse II could be harnessed to generate humanantibodies to multiple antigens, in particular, human antigens ofsignificant clinical interest.

Accordingly, individual XenoMouse II pups were challenged each with oneof three different antigen targets, human IL-8, human EGFR and humanTNF-α. Antigens were administered in two different forms, either assoluble protein, in the case of IL-8 and TNF-α or expressed on thesurface of cells (A431 cells), in the case of EGFR. For all threeantigens, ELISAs performed on sera from immunized mice indicated astrong antigen-specific human antibody (IgG, Igκ) response with titersas high as 1:3×10⁶. Negligible mouse λ response was detected.

Hybridomas were derived from spleen or lymph node tissues by standardhybridoma technology and were screened for secretion of antigen-specifichuman Mabs by ELISA.

An IL-8 immunized XenoMouse II yielded a panel of 12 hybridomas, allsecreting fully human (hIgG₂κ) Mabs specific to human IL-8. Antibodiesfrom four of these hybridomas, D1.1, K2.2, K4.2, and K4.3, were purifiedfrom ascitic fluid and evaluated for their affinity for human IL-8 andtheir potency in blocking binding of IL-8 to its receptors on humanneutrophils.

Affinity measurements were performed by solid phase measurements of bothwhole antibody and Fab fragments using surface plasmon resonance inBIAcore and in solution by radioimmunoassay (Materials and Methods). Asshown in Table V, affinity values measured for the four Mabs ranged from1.1×10⁹ to 4.8×10¹⁰ M⁻¹. While there was some variation in thetechniques employed, affinity values for all four antibodies wereconsistently higher than 10⁹ M⁻¹.

ELISA analysis confirmed that these four antibodies were specific tohuman IL-8 and did not cross-react with the closely related chemokinesMIP-1α, GROα, β, and γ, ENA-78, MCP-1, or RANTES (data not shown).Further, competition analysis on the BIAcore indicated that theantibodies recognize at least two different epitopes (data not shown).All antibodies inhibit IL-8 binding to human neutrophils as effectivelyas the murine anti-human IL-8 neutralizing antibody, whereas a controlhuman IgG₂κ antibody did not (FIG. 5A).

Fusion experiments with EGFR-immunized Xenomouse II yielded a panel of25 hybridomas, all secreting EGFR-specific human IgG₂K Mabs. Of thethirteen human Mabs analyzed, four (E2.1, E2.4, E2.5, E2.11) wereselected for their ability to compete with EGFR-specific mouse antibody225, which has previously been shown to inhibit EGF-mediated cellproliferation and tumor formation in mice (Sato et al., 1983). Thesehuman antibodies, purified from ascitic fluid, were evaluated for theiraffinity for EGFR and neutralization of EGF binding to cells. Theaffinities of these antibodies for EGFR, as determined by BIAcoremeasurements, ranged from 2.9×10⁹ to 2.9×10¹⁰ M⁻¹ (Table V).

All four anti-EGFR antibodies completely blocked EGF binding to A431cells (FIG. 5B), demonstrating their ability to neutralize its bindingto both high and low affinity receptors on these cells (Kawamoto et al.,1983). Complete inhibition of EGF binding to EGFR expressed on humanSW948 human lung carcinoma cells by all four anti-EGFR human antibodieswas also observed (data not shown). In both cases, the fully humanantibodies were as effective in inhibition of EGF binding as theanti-EGFR mouse antibody 225 and more potent than the 528 antibody (Gillet al., 1983). In both cell assays, a control human IgG₂κ antibody didnot affect EGF binding (FIG. 5B and data not shown).

Fusion experiments with TNF-α immunized Xenomouse II yielded a panel of12 human IgG₂κ antibodies. Four out of the 12 were selected for theirability to block the binding of TNF-A to its receptors on U937 cells(FIG. 5C). The affinities of these antibodies were determined to be inthe range of 1.2-3.9×10⁹ M⁻¹ (Table V).

The described Xenomouse-derived hybridomas produced antibodies atconcentrations in the range of 2-19 μg/ml in static culture conditions.Characterization of the purified antibodies on protein gels undernon-reducing conditions revealed the expected apparent molecular weightof 150 kD for the IgG₂κ antibody. Under reducing conditions the expectedapparent molecular weights of 50 kD for the heavy and 25 kD for thelight chain were detected (data not shown).

Table V, below, shows affinity constants of XenoMouse-derivedantigen-specific fully human Mabs. The affinity constants ofXenoMouse-derived human IgG₂κ Mabs specific to IL-8, EGFR, and TNF-αwere determined by BIAcore or by radioimmunoassay as described inMaterials and Methods. The values shown for IL-8 and EGFR arerepresentative of independent experiments carried out with purifiedantibodies, while the values shown for TNF-α are from experimentscarried out with hybridoma supernatants. TABLE V Human Surface Radio MabDensity Immunoassay IgG₂κ Antigen ka (M⁻¹S⁻¹) kd (S⁻¹) KA (M⁻¹) KD (M)[RU] (M⁻¹) Solid Phase Measurements Solution D1.1 IL-8 2.7 × 10⁶ 9.9 ×10⁻⁴ 2.7 × 10⁹ 3.7 × 10⁻¹⁰ 81  2.0 × 10¹⁰ D1.1 Fab IL-8 2.1 × 10⁶ 2.1 ×10⁻³ 1.1 × 10⁹ 8.8 × 10⁻¹⁰ 81  4.9 × 10¹¹ K2.2 IL-8 0.9 × 10⁶ 2.3 × 10⁻⁴4.0 × 10⁹ 2.5 × 10⁻¹⁰ 81  1.0 × 10¹⁰ K4.2 IL-8 2.5 × 10⁶ 4.1 × 10⁻⁴ 6.3× 10⁹ 1.6 × 10⁻¹⁰ 81 ND K4.3 IL-8 4.3 × 10⁶ 9.4 × 10⁻⁴ 4.5 × 10⁹ 2.2 ×10⁻¹⁰ 81  2.1 × 10¹¹ K4.3 Fab IL-8 6.0 × 10⁶ 2.1 × 10⁻³ 2.9 × 10⁹ 3.4 ×10⁻¹⁰ 81 ELISA (M) E1.1 EGFR 1.9 × 10⁶ 6.5 × 10⁻⁴ 2.9 × 10⁹ 3.46 ×10⁻¹⁰  303 1.1 × 10⁻¹⁰ E2.5 EGFR 2.1 × 10⁶ 1.8 × 10⁻⁴  1.2 × 10¹⁰ 8.44 ×10⁻¹¹  303 3.6 × 10⁻¹⁰ E2.11 EGFR 1.7 × 10⁶ 4.7 × 10⁻⁴ 3.7 × 10⁹ 2.68 ×10⁻¹⁰  303 1.1 × 10⁻¹⁰ E2.4 EGFR 2.8 × 10⁶ 9.78 × 10⁻⁵   2.9 × 10¹⁰ 3.5× 10⁻¹¹ 818 1.1 × 10⁻¹⁰ T22.1 TNF-α 1.6 × 10⁶ 1.3 × 10⁻³ 1.2 × 10⁹ 8.06× 10⁻¹⁰  107 T22.4 TNF-α 2.4 × 10⁶ 4.6 × 10⁻⁴ 5.3 × 10⁹ 1.89 × 10⁻¹⁰ 107 T22.8 TNF-α 1.7 × 10⁶ 7.5 × 10⁻⁴ 2.3 × 10⁹ 4.3 × 10⁻¹⁰ 107 T22.9TNF-α 2.3 × 10⁶ 4.9 × 10⁻⁴ 4.8 × 10⁹ 2.11 × 10⁻¹⁰  107 T22.11 TNF-α 2.9× 10⁶ 7.9 × 10⁻⁴ N/A 2.76 × 10⁻¹⁰  107

Example 10 Gene Usage and Somatic Hypermutation in Monoclonal Antibodies

The sequences of the heavy and kappa light chain transcripts from thedescribed IL-8 and EGFR-human Mabs were determined FIG. 6 and Figures [[]]. The four IL-8-specific antibodies consisted of at least threedifferent V_(H) genes (V_(H4-34)/V_(H4-21), V_(H3-30), and V_(H5-51)),four different D_(H) segments (A1/A4, K1, ir3rc, and 21-10rc) and twoJ_(H) (J_(H3) and J_(H4)) gene segments. Three different V_(κ) genes(012, 018, and B3) combined with Jκ3 and Jκ4 genes. Such diverseutilization shows that Xenomouse II is capable of producing a panel ofanti-IL-8 neutralizing antibodies with diverse variable regions.

In contrast to the IL-8 antibody transcripts, the sequences ofantibodies selected for their ability to compete with Mab 225 showedrelatively restricted V_(H) and Vκ gene usage, with three antibodies,E1.1, E2.4 and E2.5 sharing the same V_(H) gene (4-31) and E2.11containing V_(H46-61), which is highly homologous to V_(H4-31).Different D (2, A1/A4, XP1) and J_(H) (J_(H)3, J_(H)4, J_(H)5) segmentswere detected. All four antibodies were shown to share the same Vκ (018)gene. Three of them contained Jκ4, and one, E2.5, contained Jκ2.

Most V_(H) and Vκ hybridoma transcripts showed extensive nucleotidechanges (7-17) from the corresponding germline segments, whereas nomutations were detected in the constant regions. Most of the mutationsin V segments resulted in amino acid substitutions in the predictedantibody amino acid sequences (0-12 per V gene), many in CDR1 and CDR2regions (Figure _). Of note are the mutations which are shared by theheavy chain sequences of EGFR antibodies, such as the Gly→Aspsubstitution in CDR1, shared by all antibodies, or Ser→Asn substitutionin CDR2 and Val→Leu in the framework region 3 shared by threeantibodies. These results indicated that an extensive process of somatichypermutation, leading to antibody maturation and selection, isoccurring in Xenomouse II.

Discussion

This present application describes the first functional substitution ofcomplex, megabase-sized mouse loci, with human DNA fragments equivalentin size and content reconstructed on YACs. With this approach, the mousehumoral immune system was “humanized” with megabase-sized human Ig locito substantially reproduce the human antibody response in mice deficientin endogenous antibody production.

Our success in faithful reconstruction of a large portion of the humanheavy and kappa light chain loci, nearly in germline configuration,establishes YAC recombination in yeast as a powerful technology toreconstitute large, complex and unstable fragments, such as the Ig loci(Mendez et al., 1995), and manipulate them for introduction intomammalian cells. Furthermore, the successful introduction of the twolarge heavy and kappa light chain segments into the mouse germline inintact form confirms the methodology of ES cell-yeast spheroplast fusionas a reliable and efficient approach to delivering xenogeneic loci intothe mouse germline.

Characterization of Xenomouse II strains has shown that the large Igloci were capable of restoring the antibody system, comparable in itsdiversity and functionality to that of wildtype mice, and much superiorto the humoral response produced in mice bearing human Ig minigeneconstructs (Lonberg et al., 1994) or small human Ig YACs (Green et al.,1994). This difference was manifested in the levels of mature B-cells,human Ig production, class switching efficiency, diversity,preponderance of human Igκ over murine Igλ production, and magnitude ofthe human antibody response, and success in the generation of highaffinity, antigen-specific monoclonal antibodies to multiple antigens.

The levels of mature B-cells and human antibodies in Xenomouse II arethe highest yet reported for Ig-transgenic mice, representing aseveral-fold increase over the levels shown for previous mice andapproaching those of wildtype mice. In particular, the levels of thehuman IgG were more than 100 fold higher than those reported for micebearing minilocus Ig transgenes with human γ1 gene (Lonberg et al.,1994). The more efficient class switching in Xenomouse II was likely theresult of the inclusion of the entire switch regions, with all of theirregulatory elements, as well as the additional control elements on yH2,which may be important to support and maintain proper class switching.The elevated levels of mature B-cells in Xenomouse II strains are likelyto result from the higher rearrangement frequency and thus improvedB-cell development in the bone marrow due to the increased V generepertoire. B-cell reconstitution is expected to be even more pronouncedin XenoMouse II strains that are homozygous for the human heavy chainlocus.

The ratio of human κ to mouse λ light chain expression by circulatingB-cells provides a useful internal measure of the utilization of thetransgenic kappa chain locus. Whereas in mice containing one allele ofsmaller Ig YACs, an approximately equal distribution of human κ andmouse λ was observed, a significant preponderance of human κ wasdetected in Xenomouse II strains. Moreover, in animals homozygous foryK2 possessed a κ:λ ratio that is identical to wild type mice. Theseobservations together with the broad Vκ gene usage strongly suggest thatthe human proximal Vκ genes in the Xenomouse II are sufficient tosupport a diverse light chain response and are consistent with the biastoward proximal Vκ gene usage in humans (Cox et al., 1994).

Xenomouse II strains exhibited highly increased antibody diversity withV, D, and J genes across the entire span of the loci accessed by therecombination mechanism and incorporated into mature antibodies. Oncetriggered by antigen binding, extensive somatic hypermutation occurs,leading to affinity maturation of the antibodies.

The utilization pattern of V, D, J genes in Xenomouse II also indicatedthey are available and utilized in a manner reminiscent of theirutilization in humans, yielding an adult-like human antibody repertoire,which is different from the fetal-like, position-biased usage observedin Ig minigene-bearing mice (Taylor et al., 1992; Taylor et al., 1994;Tuaillon et al., 1993). The broad utilization of many of the functionalV_(H) and V_(κ) genes together with the multiplicity of antigensrecognized by the mice underscores the importance of the large V generepertoire to successfully reconstituting a functional antibodyresponse.

The ultimate test for the extent of reconstitution of the human immuneresponse in mice is the spectrum of antigens to which the mice willelicit an antibody response and the ease with which antigen-specifichigh affinity Mabs can be generated to different antigens. Unlike miceengineered with smaller human Ig YACs or minigenes, which yielded todate only a limited number of antigen-specific human Mabs (Lonberg etal., 1994; Green et al., 1994; Fishwild et al., 1996), Xenomouse IIgenerated Mabs to all human antigens tested to date. Xenomouse IIstrains mounted a strong human antibody response to different humanantigens, presented either as soluble proteins or expressed on thesurfaces of cells. Immunization with each of the three human antigenstested yielded a panel of 10-25 antigen-specific human IgG₂κ Mabs. Foreach antigen, a set of antibodies with affinities in the range of10⁹-10¹⁰ M⁻¹ was obtained. Several measures were taken to confirm thatthe affinity values represent univalent binding kinetics rather thanavidity: BIAcore assays with intact antibodies were carried out withsensor chips coated at low antigen density to minimize the probabilityof bivalent binding; for two antibodies, the assay was repeated withmonovalent Fab fragments; some of the antibodies were also tested bysolution radioimmunoassay. From the results of these measurements, weconclude that antibodies with affinities in the range of 10¹⁰ M⁻¹ arereadily attainable with the XenoMouse. The affinity values obtained forXenoMouse-derived antibodies are the highest to be reported for humanantibodies against human antigens produced from either engineered mice(Lonberg et al., Fishwild et al., 1996) or from combinatorial libraries(Vaughan et al., 1996). These high affinities combined with theextensive amino acid substitution as a result of somatic mutation in theV genes confirms that the mechanism of affinity maturation is intact inXenomouse II and comparable to that in wildtype mice.

These results show that the large antibody repertoire on the human IgYACs is being properly exploited by the mouse machinery for antibodydiversification and selection, and, due to the lack of immunologicaltolerance to human proteins, can yield high affinity antibodies againstany antigen of interest, including human antigens. The facility withwhich antibodies to human antigens can be generated by the humanimmunoglobulin genes in these mice provides further confirmation thatself tolerance at the B-cell level is acquired and not inherited.

The ability to generate high affinity fully human antibodies to humanantigens has obvious practical implications. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derivatized Mabs and thus to increase the efficacy andsafety of the administered antibodies. Xenomouse II offers theopportunity ofproviding a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated antibodyadministrations. The rapidity and reproducibility with which XenoMouseII yields a panel of fully human high affinity antibodies indicates thepotential advance it offers over other technologies for human antibodyproduction. For example, in contrast to phage display, which requiresintensive efforts to enhance the affinity of many of its derivedantibodies and yields single chain Fvs or Fabs, Xenomouse II antibodiesare high affinity fully intact immunoglobulins which can be producedfrom hybridomas without further engineering.

The strategy described here for creation of an authentic human humoralimmune system in mice can be applied towards humanization of othermulti-gene loci, such as the T cell receptor or the majorhistocompatibility complex, that govern other compartments of the mouseimmune system (Jakobovits, 1994). Such mice would be valuable forelucidating the structure-function relationships of the human loci andtheir involvement in the evolution of the immune system.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. In addition, the following references arealso incorporated by reference herein in their entirety, including thereferences cited in such references:

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1-7. (canceled)
 8. In a transgenic non-human mammal having a genome thatcomprises modifications, the modifications rendering the mammal capableof producing human immunoglobulin molecules but substantially incapableof producing functional endogenous antibody molecules, the improvementcomprising: insertion into the genome of the mammal of sufficient humanV_(H), D_(H), J_(H), Vκ, and Jκ genes such that the mammal is capableencoding greater than about 1×10⁶ different functional humanimmunoglobulin sequence combinations, without accounting for junctionaldiversity or somatic mutation events.
 9. In a transgenic non-humanmammal having a genome that comprises modifications, the modificationsrendering the mammal capable of producing human immunoglobulin moleculesbut substantially incapable of producing functional endogenous antibodymolecules, which modifications, with respect to the mammal's incapacityto produce functional endogenous antibody molecules would not allow themammal to display normal B-cell development, the improvement comprising:insertion into the genome of the mammal of sufficient human V_(H),D_(H), J_(H), Vκ, and Jκ genes such that the mammal is capable ofencoding greater than about 1×10⁶ different functional humanimmunoglobulin sequence combinations and sufficient V_(H) and Vκ genesto substantially restore normal B-cell development in the mammal.
 10. Inthe mammal of claim 9, wherein in a population of mammals B-cellfunction is reconstituted on average to greater than about 50% ascompared to wild type.
 11. A transgenic non-human mammal having agenome, the genome comprising modifications, the modificationscomprising: i. an inactivated endogenous heavy chain immunoglobulin (Ig)locus; ii. an inactivated endogenous kappa light chain Ig locus; iii. aninserted human heavy chain Ig locus, the human heavy chain Ig locuscomprising a nucleotide sequence substantially corresponding to thenucleotide sequence of yH2; and iv. an inserted human kappa light chainIg locus, the human kappa light chain Ig locus comprising a nucleotidesequence substantially corresponding to the nucleotide sequence of yK2.12. A transgenic non-human mammal having a genome, the genome comprisingmodifications, the modifications comprising: i. an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; ii. an inserted humanheavy chain Ig locus, the human heavy chain Ig locus comprising anucleotide sequence substantially corresponding to the nucleotidesequence of yH2; and iii. an inserted human kappa light chain Ig locus,the human kappa light chain Ig locus comprising a nucleotide sequencesubstantially corresponding to the nucleotide sequence of yK2.
 13. Atransgenic non-human mammal having a genome, the genome comprisingmodifications, the modifications comprising: i. an inactivatedendogenous heavy chain immunoglobulin (Ig) locus; ii. an inactivatedendogenous kappa light chain Ig locus; iii. an inserted human heavychain Ig locus, the human heavy chain Ig locus comprising a nucleotidesequence substantially corresponding to the nucleotide sequence of yH2without the presence of a human gamma-2 constant region; and iv. aninserted human kappa light chain Ig locus, the human kappa light chainIg locus comprising a nucleotide sequence substantially corresponding tothe nucleotide sequence of yK2.
 14. (canceled)
 15. A transgenicnon-human mammal having a genome, the genome comprising modifications,the modifications comprising: i. an inactivated endogenous heavy chainimmunoglobulin (Ig) locus; ii. an inserted human heavy chain Ig locus,the human heavy chain Ig locus comprising a nucleotide sequencesubstantially corresponding to the nucleotide sequence of yH2 withoutthe presence of a human gamma-2 constant region; and iii. an insertedhuman kappa light chain Ig locus, the human kappa light chain Ig locuscomprising a nucleotide sequence substantially corresponding to thenucleotide sequence of yK2. 16-25. (canceled)
 26. In a transgenicmammal, the transgenic mammal comprising a genome, the genome comprisingmodifications, the modifications comprising an inserted human heavychain immunoglobulin transgene, the improvement comprising: thetransgene comprising selected sets of human variable region genes thatenable human-like junctional diversity and human-like complementaritydetermining region 3 (CDR3) lengths.
 27. In the improvement of claim 26,wherein the human-like junctional diversity comprises average N-additionlengths of 7.7 bases.
 28. In the improvement of claim 26, wherein thehuman-like CDR3 lengths comprise between about 2 through about 25residues with an average of about 14.